Abstract: Assessing the cognitive impact of user interfaces is a shared focus of human-computer interaction researchers and cognitive scientists. Methods of cognitive assessment based on data derived from the system itself, rather than external apparatus, have the potential to be applied in a range of scenarios. The current study applied methods of analyzing kinematics to mouse movements in a computer-based task, alongside the detection response task, a standard workload measure. Sixty-five participants completed a task in which stationary stimuli were tar;geted using a mouse, with a within-subjects factor of task workload based on the number of targets to be hovered over with the mouse (one/two), and a between-subjects factor based on whether both targets (exhaustive) or just one target (minimum-time) needed to be hovered over to complete a trial when two targets were presented. Mouse movement onset times were slower and mouse movement trajectories exhibited more submovements when two targets were presented, than when one target was presented. Responses to the detection response task were also slower in this condition, indicating higher cognitive workload. However, these differences were only found for participants in the exhaustive condition, suggesting those in the minimum-time condition were not affected by the presence of the second target. Mouse movement trajectory results agreed with other measures of workload and task performance. Our findings suggest this analysis can be applied to workload assessments in real-world scenarios.