The present study aimed to develop an effective screening strategy to predict in vivo phototoxicity of multiple compounds by combined use of in vitro phototoxicity assessments and cassette dosing pharmacokinetic (PK) studies. Photochemical properties of six fluoroquinolones (FQs) were evaluated by UV spectral and reactive oxygen species (ROS) assays, and phototoxic potentials of FQs were also assessed using 3T3 neutral red uptake phototoxicity test (3T3 NRU PT) and intercalator-based photogenotoxicity (IBP) assay. Cassette dosing pharmacokinetics on FQs was conducted for calculating PK parameters and dermal distribution. All the FQs exhibited potent UV/VIS absorption and ROS generation under light exposure, suggesting potent photosensitivity of FQs. In vitro phototoxic risks of some FQs were also elucidated by 3T3 NRU PT and IBP assay. Decision matrix for phototoxicity prediction was built upon these in vitro data, taken together with outcomes from cassette dosing PK studies. According to the decision matrix, most FQs were deduced to be phototoxic, although gatifloxacin was found to be less phototoxic. These findings were in agreement with clinical observations. Combined use of in vitro photobiochemical and cassette dosing PK data will be useful for predicting in vivo phototoxic potentials of drug candidates with high productivity and reliability.