He was the younger brother to Sir Lewes Lewkenor who was also a soldier, courtier and author and served James I as his Master of the Ceremonies.
During the early 1590s, Samuel travelled on the continent "without his father’s privity or liking", visiting many of the great universities, on which he would later publish a book.
"[5] In his preface to his Discourse on Universities (1600) Samuel claimed to be "one that hath more usually been acquainted with the warlike sound of martial drums than with the schools".
He dedicated the work to his uncle, Sir Richard Lewkenor, Chief Justice of Chester and presiding judge of the Council in the Marches at Ludlow.
During the trial of the Gunpowder plotters in 1606 Lewkenor complained of jostling on the benches due to the Warden of the Fleet prison selling tickets to the spectacle, leading to overcrowding of the members.