The excavation at the Bedlam burial ground will allow construction of the eastern entrance of the new Liverpool Street Crossrail station.
A team of 60 archaeologists will work in shifts, six days a week to remove skeletons and carefully record evidence.
The work is expected to take until September when the O’Rourke team will start construction.
The Bedlam burial ground was in use from 1569 to at least 1738, spanning the start of the British Empire, civil wars, the Restoration, Shakespeare’s plays, the Great Fire of London and numerous plague outbreaks.
Jay Carver, Crossrail Lead Archaeologist said: “This excavation presents a unique opportunity to understand the lives and deaths of 16th and 17th century Londoners.
“The Bedlam burial ground spans a fascinating phase of London’s history, including the transition from the Tudor-period City into cosmopolitan early-modern London.
“This is probably the first time a sample of this size from this time period has been available for archaeologists to study in London.”