A rapid, simple and highly sensitive method was developed for the quantitative determination of lansoprazole and rabeprazole concentrations in 20 microL of human serum using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS). Analytes, along with an internal standard (lansoprazole deuterium derivatives), were separated using a mobile phase of acetonitrile/1mM ammonium formate (140/60, v/v) on a C18 analytical column and analyzed in the selected reaction-monitoring (SRM) mode. The lower limit of quantification was 0.25 ng/mL. A good linear response was observed for each analyte (from 0.25 ng to 2.5 microg/mL). This method was useful for therapeutic drug monitoring and pharmacokinetic studies.