Abstract: The detection and characterization of perphenazine metabolites in human bile were performed in this study. After collection from a psychotic patient with T-tube drainage, the bile samples were prepared by acetonitrile-induced protein precipitation and analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). According to the elemental compositions deduced from the measured accurate mass and in combination with the precursor and product ions information acquired through the MS^E function, the mass defect filter (MDF) technique and generic dealkylation tool were used to eliminate endogenous interferences and screen the metabolites. The structures of the metabolites were elucidated by comparing the fragmentation patterns between the parent drug and metabolites. After twice-daily oral administration of 4 mg of perphenazine to the patient, in addition to the parent drug, 29 metabolites, including 16 phase I and 13 phase II metabolites, were detected and characterized in the patient’s bile, 16 of which had not been previously reported. The major metabolic pathways of perphenazine in human bile include hydroxylation, desaturation, N-dealkylation, methylation, sulfation, and glucuronidation. The results can impove the metabolic pathways of perphenazine in humans.