Entrectinib (RXDX-101) is orally available inhibitor of the tyrosine kinases including tropomyosin receptor kinases (Trk) A–C, C-ros oncogene 1 (ROS1) and anaplastic lymphoma kinase (ALK), with potential antineoplastic activity. Entrectinib (ENB) granted breakthrough designation by FDA for NTRK + Solid tumors. In vitro metabolism of ENB generates quinone methide and iminium reactive intermediates that were captured by potassium cyanide and GSH, respectively forming stable conjugates that were characterized by LC–MS/MS. Seven in vitro ENB metabolites were identified through four metabolic reactions including hydroxylation, N-dealkylation, N-oxidation and reduction. Furthermore, four reactive intermediates including two quinone methide and two iminium ions were detected and the bioactivation mechanisms were supposed. In vivo metabolism of ENB was done by giving single oral dose (35.2 mg/kg) to Sprague Dawley rats. In vivo metabolism generates five phase I metabolites similar to in vitro metabolism except no metabolic reactions were identified on indazole ring. One phase II metabolite was characterized in in vivo metabolism of ENB resulted from glucuronidation of hydroxyl metabolite of ENB. Reporting these data for ENB is very crucial in the development stage. Reviewing literatures revealed the absence of previous articles have been done for the ENB in vitro or in vivo metabolism study or structural characterization of the formed reactive intermediates.