Walsingham, who had long been on the watch, was now in possession of all the evidence that he was likely to get, for Babington soon discovered that he had been betrayed by somebody, whom he could not tell; and though he remained in London as though there were no danger, he made preparations for the escape of Ballard to the Continent, by procuring him a passport under a feigned name. Every moment might throw fresh light on the deception, and allow the escape of the victims. On the 4th of August, therefore, Babington found his house entered by the pursuivants of Walsingham, and Ballard, who had not got off, was there seized. Babington escaped for the moment, but was arrested on the 7th, and was taken to the country house of Walsingham, but escaped from the servants into whose charge he was given. With his friends and accomplices, Gage, Charnock, Barnewell, and Donne, he concealed himself in St. John's Wood, till they were compelled by hunger to make their way to the house of their common friend Bellamy, at Harrow, who concealed them in his outhouses and gardens. But the cunning Walsingham had his agents on their trail the whole time, and on the 15th they walked into the premises of Bellamy, secured the concealed conspirators, together with their host, his wife and brother, and conveyed them, amid the shouts and execrations of the populace, and the universal ringing of bells, to the Tower, whither also were soon brought Abingdon, Tichbourne, Tilney, Travers; the only one of the friends of Babington that escaped being Edward Windsor, the brother of Lord Windsor.

On the 13th of September, Babington, Ballard, Savage, Donne, Barnewell, and Tichbourne were put upon their trial, charged with a conspiracy to murder Elizabeth, and raise a rebellion in favour of the Queen of Scots. They pleaded guilty to one or other of the charges, and seven others pleaded not guilty; but all were alike convicted, and condemned to the death of traitors. The greater part of them appear to have taken no part in the blacker part of the conspiracy, the design to murder Elizabeth; and some of them, as Tichbourne and Jones, declared that they had taken no part whatever, but merely kept the secret for the sake of their friends. Bellamy was condemned for affording them an asylum; his wife escaped through a flaw in the indictment. Pooley, the decoy, was imprisoned as a blind, and then liberated; and Gifford was already in prison in Paris, where, three years later, he died. On the 20th and 21st of September, 1586, they were executed in Lincoln's Inn Fields, because they used there to hold their meetings.

Though no mention was made on the trial of any participation of the Queen of Scots in this conspiracy, nothing was farther from the intention of Elizabeth and her ministers than her escape. They had already prepared for her death by the bill passed empowering twenty-four or more of the Lords of the Council and other peers to sit in judgment on any one concerned in attempts to raise rebellion, or to injure the queen's person. To procure every possible evidence for this end the following stratagem was used:—The Queen of Scots was kept in total ignorance of the seizure of the conspirators, and on the copy of her letter to Babington being laid before the Council, an order was sent down to Sir Amyas Paulet to seize all her papers and keep her in more rigorous confinement. Accordingly, one morning, Mary took a drive in her carriage, accompanied, as was her custom, by Paulet, but with a larger attendance. When Mary desired to return, Paulet told her that he had orders to convey her to Tixall, a house belonging to Sir Walter Aston, about three miles distant. Astonished and alarmed, Mary refused to go, and declared that if they took her there it should be by force. She must have suspected the design of searching her cabinets during her absence; but, in spite of her protestations and her tears, she was compelled to proceed. There she was confined to two rooms only, was guarded in the strictest manner, and debarred the use of pen, ink, and paper. Meanwhile Sir William Wade arrived at Chartley, and proceeded to break open the cabinets and take possession of all her letters and papers, as well as those of her secretaries. A large chest was filled with these papers, amongst which were Mary's own minute of the answer to Babington, and other damning proofs. It was determined to bring her to a public trial.

Mary was now removed to Fotheringay Castle in Northamptonshire, in preparation for her trial. It was first proposed to convey her to the Tower, but they feared her friends in the City; then the castle of Hertford was suggested, but that also was thought too near the capital; and Grafton, Woodstock, Coventry, Northampton, and Huntingdon were all proposed and rejected, showing that her enemies were well aware of the seriousness of the business they contemplated.

On the 5th of October a commission was issued to forty-six persons, peers, privy councillors, and judges, constituting a court competent to inquire into and determine all offences committed against the statute of the 27th of the Queen, either by Mary, daughter and heiress of James V., late of Scotland, or by any other person whomsoever. The moment this was known, Chasteauneuf, the French ambassador, demanded in the name of his sovereign that Mary should be allowed counsel, according to the universal practice of civilised nations. But Elizabeth sent him a message by Hatton, that "she did not require the advice or schooling of foreign powers to instruct her how she ought to act."

On the 12th the commissioners arrived at the castle. They were the Lord Chancellor Bromley, the Lord Treasurer Burleigh, and many other magnates.

On the 14th the Court assembled in the great hall of Fotheringay, at the upper end of which was placed a chair of State with a canopy, as for the queen of England; and below it, at some distance, a chair without a canopy, for the Queen of Scots. The Chancellor, Bromley, opened the Court by informing Mary that the Queen of England, having heard that she had conspired against her state and person, had deputed them to inquire into the fact. Upon this Mary, who had at first refused to plead at all, entered her solemn protest against their authority, declaring that she had come as a friendly sovereign to seek aid from her cousin, the Queen of England, and had been unjustly detained by her as a prisoner; on that ground she denied their authority to try her. It was permitted to record her protest, together with the Chancellor's reply.

The charges against her were two: first, that she had conspired with traitors and foreigners to invade the realm, and secondly, to compass the death of the queen. As to the first charge Mary pleaded guilty to it, and justified it. They grounded this charge on a host of letters intercepted or found in her cabinets, to and from Mendoza, Paget, Morgan, and others. From these it appeared that she had sanctioned an invasion on her behalf, had offered to raise her friends to support it, and had requested that those in Scotland should make themselves master of the person of her son, and prevent any aid from being sent to the Government of England. When they came to the second charge, the conspiracy to murder Elizabeth, she denied any participation in it totally, indignantly, and with tears. She called God to witness the truth of her assertion, and prayed Him, if she were guilty of such a crime, to grant her no mercy. The proofs produced to establish her approval of this design were—first, the copy of the letter of Babington, in which was this passage:—"For the dispatch of the usurper, from the obedience of whom, by the excommunication of her, we are made free, there be six noble gentlemen, all my private friends, who, for the zeal they bear to the Catholic cause and your majesty's service, will undertake the tragical execution." Next there was a copy of seven points, which were professedly derived from her answer to Babington, the sixth of which was, "By what means do the six gentlemen deliberate to proceed?" After these came the confessions of Mary's secretaries, and, finally, reported admissions in her letters to her foreign correspondents of having received these intimations of their intention to assassinate the queen, and of having given them cautions and instructions on this point.

Mary at first denied any correspondence with Babington, but she soon saw enough to convince her that they had the correspondence in their possession, and admitted having written the note of the 18th, but not any such answer to Babington on the 17th of July, as they asserted. She demanded the production of the original letters, and the production of her secretaries Nau and Curle face to face with her, for that Nau was timid and simple, and Curle so accustomed to obey Nau, that he would not do otherwise; but she was sure that in her presence they would not venture to speak falsely. But neither of these things, no doubt for the strongest of reasons, was consented to. As to her letters, she said it was not the first time that they had been garbled and interpolated. It was easy for one man to imitate the writing and ciphers of another; and she greatly feared that Walsingham had done it in this instance, to practise against the lives of both herself and her son. In fact, her defence was most ingenious but quite unconvincing.