He was the son of Sir John Throckmorton and a nephew of Sir Nicholas Throckmorton, one of Elizabeth's diplomats. Sir John had held the post of Chief Justice of Chester but was removed in 1579, a year before his death. The reasons for Sir John's removal from the bench are unclear; he may have been guilty of abuses in the administration of justice, but he may also have been singled out for punishment for his pro-Catholic beliefs.
Throckmorton was educated in Oxford and entered the Inner Temple in London as a pupil in 1576. In 1580, he traveled to the European continent and met leading Catholic malcontents from England in Spain and France. After his return to England in 1583, he served as an intermediary for communications between supporters of the Catholic cause on the continent, the imprisoned Mary, Queen of Scots, and the Spanish ambassador Bernardino de Mendoza.
Throckmorton's activities raised the suspicions of Sir Francis Walsingham, Elizabeth I's spymaster. A search of his house produced incriminating evidence and, after torture on the rack, Throckmorton confessed his involvement in a plot to overthrow the Queen and restore the Catholic Church in England. An invasion led by Henry I, Duke of Guise would have been coupled with an orchestrated uprising of Catholics within the country.