We evaluated four new molecular targeting agents using our cell line panel with 17 biliary tract carcinoma cell lines. PI3K/mTOR inhibitor showed promising activity in many cell lines including gemcitabine-insensitive ones. Abstract Biliary tract carcinoma (BTC) is an extremely malignant tumor, but available treatment options are limited. Despite of needs for novel therapies, few BTC-related resources are currently available for evaluation of candidate drugs. To address this issue, we have recently established 13 cell lines from surgical specimens from Japanese BTC patients. In the present study, we evaluated four new molecular targeting agents using our BTC cell-based assay panel with 17 BTC cell lines. PI3K/mTOR dual inhibitor LY3023414 showed activity at submicromolar concentration ranges against 13 of the 17 cell lines tested, including the ones with gemcitabine insensitivity. In conclusion, we demonstrated that in vitro study with the BTC cell line panel would be an efficient approach to screen for novel therapeutic strategies. Although this is preliminary result and further investigations are required for confirmation, PI3K/mTOR inhibitor might be a potential target for BTC drug development.