Phototoxicity is an acute toxic response induced by skin irradiation after the systemic or local administration of a chemical and subsequent exposure to light. Thus, it is necessary to evaluate phototoxicity of chemicals or products that are applied to the skin. Although the previous studies on the assessment of phototoxicity have been done using animals, it is necessary to establish alternative test methods, as EU ban animal-tested cosmetic ingredients or products. In vitro 3T3 NRU PT is an alternative method for identification of phototoxicity hazard chemicals as it compares cytotoxicity between UV irradiation and non-irradiation groups. Accordingly the accumulated information of phototoxicity assessment and its data has been found to be insufficient in Korea. Thus, we tested ten phototoxic and three non-phototoxic chemicals, and assessed two UV filter ingredients using in vitro 3T3 NRU PT mehtod. As a result, we identified that this method can be used to predict phototoxicity of chemicals. However, Eusolex 9020, UVA filter ingredient, was misclassified in vitro as a false positive compared with in vivo result. It suggests that other adequate test methods should be accompanied in order to enhance accuracy of phototoxicity prediction.