Neurobiological research in genetically tractable organisms relies heavily on robust assays for behavioral phenotypes. The simple body plan of the nematode Caenorhabditis elegans makes it particularly amenable to the use of automated microscopy and image analysis to describe behavioral patterns quantitatively. Forward genetic screens and screens of drug libraries require high-throughput phenotyping, a task traditionally incompatible with manual scoring of quantitatively varying behaviors. High-throughput automated analysis of C. elegans movement behavior is now possible with several different tracking software packages. The Multiworm Tracker (MWT) described here is designed for high-throughput analysis: it can record dozens of worms simultaneously at 30 frames per second for hours or days at a time. This is accomplished by performing all image analysis in real time, saving only the worm centroid, bearing, and outline data to the disk. To simplify image processing, the system focuses only on worms that have moved, and detects and discards worms that are touching rather than trying to isolate them computationally. Because the software is entirely automated, protocols can run unattended once the worms have been placed and the software has been started. The MWT does not save images for later analysis, but behavior can be validated manually with a companion analysis tool that replays recorded body postures. This protocol describes a basic basal movement assay on food using the MWT; similar protocols apply to related assays and to similar multiple animal trackers. The protocol can be extended to a variety of assays ranging from tap response to chemotaxis.