Abstract Introduction A series of experiments were undertaken to evaluate the accuracy, precision, specificity, and sensitivity of an automated, infrared photo beam-based open field motor activity system, the MotorMonitor v. 4.01, Hamilton-Kinder, LLC, for use in a good laboratory practices (GLP) Safety Pharmacology laboratory. Methods This evaluation consisted of two phases: (1) system validation, employing known inputs using the EM-100 Controller Photo Beam Validation System, a robotically controlled vehicle representing a rodent and (2) biologic validation, employing groups of rats treated with the standard pharmacologic agents diazepam or d-amphetamine. The MotorMonitor's parameters that described the open-field activity of a subject were: basic movements, total distance, fine movements, x/ y horizontal ambulations, rearing, and total rest time. These measurements were evaluated over a number of zones within each enclosure. Results System validation with the EM-100 Controller Photo Beam Validation System showed that all the parameters accurately and precisely measured what they were intended to measure, with the exception of fine movements and x/ y ambulations. Biologic validation using the central nervous system depressant diazepam at 1, 2, or 5 mg/kg, i.p. produced the expected dose-dependent reduction in rat motor activity. In contrast, the central nervous system stimulant d-amphetamine produced the expected increases in rat motor activity at 0.1 and 1 mg/kg, i.p, demonstrating the specificity and sensitivity of the system. Discussion Taken together, these studies of the accuracy, precision, specificity, and sensitivity show the importance of both system and biologic validation in the evaluation of an automated open field motor activity system for use in a GLP compliant laboratory.