Adult behavioral assays have been used with great success in Drosophila melanogaster to identify circadian rhythm genes. In particular, the locomotor activity assay can identify altered behavior patterns over the course of several days in small populations, or even individual flies. Commercially available, highly efficient automated systems allow for continuous data collection from large numbers of individuals, and analytical tools make it possible to quickly analyze multiple aspects of circadian behavior from each experiment. These features make the locomotor activity assay useful for highthroughput analyses, leading to the rapid discovery and functional characterization of many Drosophila circadian rhythm genes. The locomotor assay described here can simultaneously assess both circadian and sleep behavior, and several methods can be used to analyze the data generated from such assays. This protocol details the use of the Drosophila Activity Monitoring (DAM) System from TriKinetics. Briefly, the system records activity from individual flies maintained in sealed tubes placed in activity monitors. An infrared beam directed through the midpoint of each tube measures an “activity event” each time a fly crosses the beam. Events detected over the course of each consecutive sampling interval are summed and recorded over the course of the experiment for each fly. The general approaches described here can be applied to a wide range of behavioral activity experiments, including sleep deprivation analyses and general studies of hypoactivity and hyperactivity.