Sapitinib (AZD8931, SAP) is an epidermal growth factor receptor (EGFR) family (pan-erbB) tyrosine kinase inhibitor. In multiple tumor cell lines, SAP has been shown to be a much more potent inhibitor of EGF-driven cellular proliferation than gefitinib. In this in vitro metabolic study, we tested the generation of reactive intermediates from SAP using human liver microsomes and a capturing agent (potassium cyanide) to trap the iminium reactive intermediates. The same metabolic reaction was further repeated in the presence of methoxyamine to trap aldehyde intermediates. The identification of SAP metabolites revealed that the hydroxylation metabolic reaction represents the major in vitro metabolic pathway occurring at the piperidine moiety. We characterized six in vitro phase I metabolites in addition to three reactive intermediates (i.e., two iminiums and one aldehyde), therefore suggesting two probable SAP-bioactivation pathways. We hypothesized that the piperidine ring nitrogen (cyclic tertiary amine) activated the two adjacent α-carbons within the ring. The oxidative dealkylation of the N-acetamide group led to an unstable aldehyde that was trapped using methoxyamine, generating an oxime adduct that was detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To the best of our knowledge, this is the first study presenting the structural characterization of SAP reactive intermediates.