Recently, attention has been drawn to drug-induced phototoxic skin responses, and avoidance of this undesired side effect is necessary for pharmaceutical development. We previously proposed that determination of reactive oxygen species (ROS) generated from photoirradiated compounds would be effective for the prediction of the phototoxic potential. In this investigation, a high-throughput ROS assay system was developed using a multiwell plate and quartz reaction container. The experimental conditions of irradiance uniformity, UV intensity, exposure time, temperature and solvent systems were found to affect the generation of ROS, and thus the conditions of the ROS assay were optimized. The intra- and inter-day R.S.D. values for the determination of ROS from quinine (200 microM) irradiated at 250 W/m(2) for 1h was found to be less than 3.3 and 4.5%, respectively. The results from the ROS assay of 39 compounds allowed us to estimate classification criteria to identify the ability of phototoxic/photochemical responses. The developed assay system will be an effective tool for predicting the phototoxic potential of pharmaceutical candidates in early stage of pharmaceutical development.