In this unit, we describe an inexpensive and versatile method for optogenetic stimulation of a large population of genetically engineered Caenorhabditis elegans worms while quantitatively analyzing behavior. A custom light-emitting diode light source is used to deliver blue-light stimuli, causing direct depolarization of neurons expressing the light-gated cation channel Channelrhodopsin-2, which in turn evokes behavioral responses. The behavioral responses are recorded by a high-throughput machine vision-based tracking system, the Multi-Worm Tracker, for detailed analysis. This approach allows researchers to bypass technical obstacles to simultaneously deliver uniform stimuli to a large number of freely behaving animals and investigate the neural underpinnings of behavior. © 2018 by John Wiley & Sons, Inc.