A sensitive high-performance liquid chromatography positive ion atmospheric pressure chemical ionization tandem mass spectrometry method was developed and validated for the quantification of pregabalin in human plasma. Following liquid-liquid extraction, the analyte was separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 160-142 for pregabalin and m/z 482-258 for the internal standard. The assay exhibited a linear dynamic range of 1-10,000ng/mL for pregabalin in human plasma. The lower limit of quantification was 1ng/mL with a relative standard deviation of less than 11.4%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 4.0min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic studies.