Loxapine is an antipsychotic medication used for the treatment of schizophrenia. In vivo, loxapine is metabolized to multiple metabolites. A high performance liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of loxapine and 4 of its metabolites, loxapine N-oxide, amoxapine (N-desmethyl loxapine), 8-hydroxyloxapine and 7-hydroxyloxapine, in human plasma to support regulated clinical development. During method development, several technical challenges such as poor chromatography, separation of structural isomers, and inadequate sensitivity were met and overcome. The final method utilized micro-elution solid phase extraction (SPE) to extract plasma samples (100µL), and the resulting extracts were analyzed using reversed phase LC-MS/MS using a turbo-ionspray interface in positive ionization mode with selected reaction monitoring (SRM). The method was fully validated according to the current regulatory guidance for bioanalysis over the calibration curve range 0.0500-50.0ng/mL for all analytes using 1/x2-weighted linear regression analysis. Based on three separate runs, the between-run precision and inter-day precision for all five analytes at all concentrations, including the LLOQ (lower limit of quantitation) quality control at 0.0500ng/mL, varied from 0.0% to 13.8%, while the accuracy ranged from 86.4% to 109.3% of nominal. The extraction recoveries of loxapine and the four metabolites were above 80%. Various forms of short-term and long-term stability were established in both solutions and matrix, including the stability of loxapine and the four metabolites in human plasma for up to 260days of storage at -20°C. This method has been used to support a regulated clinical study, which included the successful execution of incurred sample reanalysis (ISR) testing. To the best of our knowledge, this is the first published methodology in which these five analytes were quantified with a single extraction and injection.