A robust and highly sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of pericyazine in human plasma. The plasma sample was alkalized with sodium hydroxide solution and handled by liquid-liquid extraction with ethyl acetate after adding perphenazine as an internal standard (IS). The analytes were separated on an Ultimate™ AQ-C18 analytical column at 40 °C, with a gradient elution consisting of A (aqueous phase: 5 mM ammonium acetate buffer solution containing 0.1% formic acid) and B (organic phase: acetonitrile) at a flow rate of 0.350 mL/min. The detection was conducted on an API 4000 tandem mass spectrometer coupled with electrospray ionization (ESI) source in positive ion mode. The multiple reaction monitoring (MRM) transitions, m/z 366.5 > 142.4 for pericyazine, m/z 382.5 > 142.4 for its 7-hydroxy and sulphoxide metabolites and m/z 404.3 > 171.3 for IS were chosen to achieve high selectivity in the simultaneous analyses. The method exhibited great improvement in sensitivity (LLOQ of 0.021 ng/mL) and good linearity over the concentration range of 0.021–9.90 ng/mL. The intra- and inter-day precision, accuracy, and stability results were within the acceptable limits and no matrix effect was observed. This method was successfully applied in a bioequivalence study to evaluate the pharmacokinetics in 20 healthy male Chinese volunteers. Additional exploratory analyses of 7-hydroxy and sulphoxide metabolites of pericyazine in the same samples suggest that the unchanged drug is predominant in the plasma and suitable for the bioequivalence comparison after a single oral administration of 10 mg pericyazine.