Objective: To develop and validate a sensitive high performance liquid chromatography-tandem mass spectrometry method for the simultaneous determination of clopidogrel and its metabolites and subsequently to use it to investigate pharmacokinetic properties of clopidogrel in mice. Methods: Clopidogrel and its metabolites were determined at an electron spray ionization (ESI) source using multiple reaction monitoring (MRM) in positive ion mode after protein precipitation by acetonitrile，in which piroxicam was added as an internal standard. Chromatographic separation was achieved on a Poroshell 120 SB-C18 column (100×2.1 mm, 2.7 μm, Agilent Technologies) using a mobile phase that consisted of both solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) in a gradient elution manner. The pharmacokinetics of clopidogrel was investigated in mice receiving a single dose of clopidogrel (10 mg/kg) by gavage administration. Results: The simultaneous assay of clopidogrel and its metabolites achieved good linearity (r> 0.99) within the range of the spiked plasma concentrations. The recoveries were greater than 80%, and no significant matrix effect was found. The both intra- and inter-day RSDs were less than 15%, and the accuracy ranged from -2.40% to 5.00%. The stability of the method was good. In addition, a series of pharmacokinetics parameters were obtained in mice after receiving a single oral dose of clopidogrel through this analytical method. Conclusion: The modified methodology was highly sensitive, accurate, and reproducible for the simultaneous determination of clopidogrel and its three major metabolites in mouse plasma and therefore, it was successfully applied in pharmacokinetic studies of clopidogrel in mice.