Leiden flourished in the 16th and 17th century. At the close of the 15th century the weaving establishments (mainly broadcloth) of Leiden were very important. After the expulsion of the Spaniards, Leiden cloth, Leiden baize and Leiden camlet were familiar terms. In the same period, Leiden developed an important printing and publishing industry. 

In 1572, the city sided with the Dutch Revolt against Spanish rule and played an important role in the Eighty Years War. Besieged from May until October 1574 by the Spanish, Leiden was relieved by the cutting of the dikes, thus enabling ships to carry provisions to the inhabitants of the flooded town. As a reward for the heroic defence of the previous year, the University of Leiden was founded by William I of Orange in 1575. Yearly on 3 October, the end of the siege is still celebrated in Leiden. 

Leiden is also known as the place where the Separatists (as well as some of the first settlers of New Amsterdam) lived (and operated a printing press) for a time in the early 17th century before their departure to Massachusetts and New Amsterdam in the New World.