Abstract: A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method is developed and validated for determination of clenbuterol in human plasma. The analyte is extracted from plasma samples by liquid-liquid extraction, separated through a Zorbax XDB C8 column and detected by tandem mass spectrometry with an electrospray ionization interface. Diphenhydramine is used as the internal standard. The method has a lower limit of quantitation (LOQ) of 10.0 ng/L for clenbuterol. The intra- and inter-run precision is measured to be below 7.2%. The inter-run accuracy is within ±2.5% for the analyte. The chromatographic run time is approximately 3.2 min. More than 120 samples can be assayed daily with this method, including sample preparation, data acquisition and processing. The method is applied for the evaluation of the pharmacokinetics of clenbuterol in 20 volunteers after an oral dose of 80 μg clenbuterol hydrochloride.