Henslowe was originally a dyer by trade, and was unusual among theatre managers of the day in never having been an actor. He built the Rose Theatre in Southwark in 1587, presumably as an extension of his earlier financial enterprises, which included starch making and money-lending. His career as a theatre manager appears to have taken off in 1592, when his stepdaughter married Edward Alleyn the actor. Henslowe's primary connection to his theatres was financial and administrative rather than artistic; he took a percentage of the ticket sales, and his account books for 1593-1603 (known as his 'Diary') also record payments for licences, costumes, and to playwrights. His 'Inventory' (of costumes, properties etc.) of 1598 also survives.

Alleyn was born in the parish of St Botolph, Bishopsgate, on 1 Sept. 1566. He was well-established as an actor by 1592, and regarded as a leading actor (and even a celebrity) by the middle of the decade. He married Joan Woodward, step-daughter to Henslowe, in 1592. He seems increasingly to have become an entrepreneur rather than a player; in particular, he took a complete break from playing between 1597-1600, and seems to have ceased playing completely in about 1604: his last recorded role was as the Genius of the City in the Magnificent Entertainment for King James (1604), and Heywood's Apology for Actors ( c . 1608) describes him as recently retired. Alleyn founded Dulwich College, as the 'College of God's Gift', in 1619. He died in 1626.