Phototoxic properties of systemically applied pharmaceuticals may be the cause of serious adverse drug reactions. Therefore, a reliable preclinical photosafety assessment strategy, combiningin vitroandin vivoapproaches in a quantitative manner, is important and has not been described so far. Here, we report the establishment of an optimized modified murine local lymph node assay (LLNA), adapted for phototoxicity assessment of systemically applied compounds, as well as the test results for 34 drug candidates in thisin vivophoto-LLNA. The drug candidates were selected based on their ability to absorb ultraviolet/visible light and the photo irritation factors (PIFs) determined in the well-establishedin vitro3T3 neutral red uptake phototoxicity test. Anin vivophototoxic potential was identified for 13 of these drug candidates. The use of multiple dose levels in the described murinein vivophototoxicity studies enabled the establishment of no- and/or lowest-observed-adverse-effect levels (NOAELs/LOAELs), also supporting human photosafety assessment. Anin vitro-in vivocorrelation demonstrated that a drug candidate classified as “phototoxic”in vitrois not necessarily phototoxicin vivo. However, the probability for a drug candidate to cause phototoxicityin vivoclearly correlated with the magnitude of the phototoxicity identifiedin vitro.