Recent advances in imaging allow to monitor in real time the behaviour of individuals under a given stress. Light is a common stressor that alters the behaviour of fish larvae and many aquatic invertebrate species. The water flea Daphnia magna exhibits a vertical negative phototaxis, swimming against light trying to avoid fish predation. The aim of this study was to develop a high-throughput image analysis system to study changes in the vertical negative phototaxis of D. magna first reproductive adult females exposed to 0.1 and 1 μg/L of four neuro-active drugs: diazepam, fluoxetine, propranolol and carbamazepine. Experiments were conducted using a custom designed experimental chamber containing four independent arenas and infrared illumination. The apical-located visible light and the GigE camera located in front of the arenas were controlled by the Ethovision XT 11.5 sofware (Noldus Information Technology, Leesburg, VA). Total distance moved, time spent per zone (bottom vs upper zones) and distance among individuals were analyzed in dark and light conditions, and the effect of different intensities of the apical-located visible light was also investigated. Results indicated that light intensity increased the locomotor activity and low light intensities allowed to better discriminate individual responses to the studied drugs. The four tested drugs decreased the response of exposed organisms to light: individuals moved less, were closer to the bottom and at low light intensities were closer each other. At high light intensities, however, exposed individuals were less aggregated. Propranolol, carbamazepine and fluoxetine induced the most severe behavioural effects. The tested drugs at environmental relevant concentrations altered locomotor activity, geotaxis, phototaxis and aggregation in D. magna individuals in the lab. Therefore the new image analysis system presented here was proven to be sensitive and versatile enough to detect changes in diel vertical migration across light intensities and low concentration levels of neuro-active drugs.