ObjectiveThe ability to accurately and efficiently quantify mouse locomotor activity is essential for evaluating therapeutic efficacy and phenotyping genetically modified mice, in particular for the research of neuromuscular diseases. Our objective is to develop a program for video tracking of mice and locomotion analysis.ResultsHere we describe a MATLAB program for video tracking of mice and locomotion analysis. The system is composed of a webcam, an open field, and a computer with MATLAB installed. Animal behavior is recorded by the webcam and the video is then analyzed for mouse position on each frame by a customized MATLAB code. The system has been tested for analyzing two or more mice simultaneously placed in individual chambers. The accumulative moving distance, velocity and thigmotaxis (percentage of time spending in the outer peripheral of the arena, which is commonly used as an index of anxiety) within a test period can be readily obtained. This system can be easily implemented in any laboratory as an in vivo locomotion assay to assess the neuromuscular abnormality of genetically modified animals and the impact of therapeutic interventions.