There is an unmet need to develop novel, effective medical therapies for cholangiocarcinoma (CCA). The Hippo pathway effector, YAP, is oncogenic in cholangiocarcinoma; however, attempts to therapeutically target YAP in vivo have been unsuccessful thus far. Recently, we described a novel role for the Src-family kinase LCK in activating YAP through tyrosine phosphorylation. This led to the hypothesis that LCK may be a viable therapeutic target in cholangiocarcinoma to regulate YAP activity. NTRC 0652-0 is a novel tyrosine kinase inhibitor with specificity for LCK. NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in cholangiocarcinoma cells. NTRC 0652-0 treatment led to decreased tyrosine phosphorylation of YAP, inhibition of its activity and apoptotic cell death in cholangiocarcinoma cell lines, associated with inhibition of MCL1 expression - consider rephrasing this last part to make the sentence read better. The extent of sensitivity of patient-derived organoids to NTRC 0652-0 correlated with basal YAP tyrosine phosphorylation and suppression of YAP co-transcriptional activity following drug treatment. In a patient-derived xenograft CCA model bearing an FGFR2 fusion, daily oral treatment with NTRC 0652-0 produced stable tumor drug levels, acceptable toxicity, and significantly decreased tumor growth. Overall, a novel LCK inhibitor, NTRC 0652-0, inhibited YAP signaling and demonstrated preclinical efficacy in CCA cell-lines, organoids, and patient derived xenograft models.