Abemaciclib (Verzenio®) is approved as tyrosine kinase inhibitor (TKI) for breast cancer treatment. In this study, in vitro phase I metabolic profiling of Abemaciclib (ABC) was done using rat liver microsomes (RLMs). We checked the formation of reactive intermediates in ABC metabolism using (RLMs) in the presence of potassium cyanide (KCN) that was used as capturing agent for iminium reactive intermediates forming a stable complex that can be characterized by LC-MS/MS. Nine in vitro phase I metabolites and three cyano adducts were identified. The metabolic reactions involved in formation of these metabolites and adducts are reduction, oxidation, hydroxylation and cyanide addition. The bioactivation pathway was also proposed. Knowing the electrodeficient bioactive centre in ABC structure helped in making targeted modifications to improve its safty and retain its efficacy. Blocking or isosteric replacement of α carbon to the tertiary nitrogen atoms of piperazine ring can aid in reducing toxic side effects of ABC. No previous articles were found about in vitro metabolic profiling for ABC or structural identification of the formed reactive metabolites for ABC.