During the development of a method for quantitative determination of venlafaxine and its major metabolite O-desmethylvenlafaxine, elevated concentrations of the analyte as well as co-eluting matrix compounds caused ion suppression. This ion suppression was inconsistent and therefore influenced the reproducibility of detection. The use of atmospheric pressure photoionization (APPI) in the positive mode was investigated as a tool to circumvent this problem. Employing APPI resulted in negligible ion suppression and increased linearity of the concentration range. A selective, sensitive and rapid liquid chromatography/tandem mass spectrometry method for the determination of venlafaxine and its major metabolite O-desmethylvenlafaxine in human plasma was developed. The analyte was extracted from plasma into tert-butyl methyl ether followed by back extraction into 2% formic acid. An Agilent 1100 high-performance liquid chromatography (HPLC) system, employing reversed-phase chromatography on a cyano column, coupled to an Applied Biosystems API 3000 triple quadrupole mass spectrometer set to multiple reaction monitoring (MRM) mode, was used for separation and detection of the analytes. The method was validated between 2.36-605 ng per mL with a mean recovery of approximately 88% for both parent compound and metabolite analytes. APPI technology was employed to improve the reproducibility of detection enabling rapid, selective and sensitive quantification of venlafaxine and O-desmethylvenlafaxine in human plasma samples.