Copanlisib (CNB; Aliqopa™) is a novel, intravenous phosphoinositide 3-kinase inhibitor used to treat various solid and hematological malignancies. CNB was recently approved by the U.S. FDA to treat adults that relapsed after two preceding systemic therapies. Using LC-MS/MS, we screened for the in vitro metabolites of CNB formed in human liver microsomes (HLMs) and probed for the generation of reactive electrophiles using methoxyamine and potassium cyanide as nucleophiles to capture reactive electrophiles by forming stable adducts that are suitable for identification by LC-MS/MS. Seven CNB phase I metabolites generated by oxidation, hydroxylation, oxidative dealkylation, reduction, and N-oxidation were identified. In addition, four reactive electrophiles, 2 aldehydes and 2 iminium ions, were identified, and a prediction of the corresponding bioactivation mechanism is presented. The formation of reactive metabolites may be associated with the side effects reported for CNB. To our knowledge, this is the first report on the detailed structural characterization of reactive intermediates generated in CNB metabolism.