<p>Phototoxicity is elicited by topically or systemically administered photoreactive chemicals after the exposure of tissues such as skin and eyes to light. The in vitro 3T3 phototoxicity test (OECD TG432), which has been well established, could lead to false-negative results for chemicals predominantly absorbing in the ultraviolet B (UVB) range, since UVB itself is cytotoxic to 3T3 cells and only ultraviolet A (UVA) and visible light irradiation is used. On the other hand, the ROS assay (OECD TG495), which evaluates photoreactivity without using cells, is a test that can use UVB irradiation. However, it is known that this assay shows a high incidence of false-positive results. Therefore, we developed a novel in vitro UVB-induced phototoxicity test method using human ES/iPS cells-derived retinal pigment epithelial cells (RPEs). </p><p> To assess UVB tolerance, RPEs and 3T3 cells were irradiated with UVB and cell viability was measured. As a result, RPEs were found to be more UVB tolerant than 3T3 cells. RPEs were then exposed to a phototoxic substance predominantly absorbing in the UVB range / non-phototoxic substance, followed by UVB irradiation. Cell viability was measured to evaluate phototoxicity. The results were obtained as expected and the newly developed test method predicted more accurately than the existing methods (TG432 and TG495). These results suggest that this method can be useful for phototoxic evaluation of UVB absorbers.</p>