Currently, measuring ethanol behaviors in flies depends on expensive image analysis software or time intensive experimental observation. We have designed an automated system for the collection and analysis of locomotor behavior data, using the IEEE 1394 acquisition program dvgrab, the image toolkit ImageMagick and the programming language Perl. In the proposed method, flies are placed in a clear container and a computer-controlled camera takes pictures at regular intervals. Digital subtraction removes the background and non-moving flies, leaving white pixels where movement has occurred. These pixels are tallied, giving a value that corresponds to the number of animals that have moved between images. Perl scripts automate these processes, allowing compatibility with high-throughput genetic screens. Four experiments demonstrate the utility of this method, the first showing heat-induced locomotor changes, the second showing tolerance to ethanol in a climbing assay, the third showing tolerance to ethanol by scoring the recovery of individual flies, and the fourth showing a mouse’s preference for a novel object. Our lab will use this method to conduct a genetic screen for ethanol-induced hyperactivity and sedation, however, it could also be used to analyze locomotor behavior of any organism.