Abstract: In this paper, we developed an accurate and sensitive LC-MS/MS method for the determination of amoxicillin and clavulanic acid in human plasma. A 50% aqueous acetic acid solution was used as a stabilizer, and the plasma samples were evaporated to dryness and resolved after protein precipitation on ice bathing and then were placed in an autosampler for injection. The gradient was eluted by Hedera ODS-2 column(2. 1 mm×150 mm). The aqueous phase was an aqueous solution containing 0. 2% acetic acid. The organic phase was methanol. The amoxicillin and clavulanic acid were detected under negative ion detection with electrospray ionization(ESI)in multiple reaction monitoring(MRM)mode of m/z 364. 1→223. 1 and 198. 1→135. 9 in the triple quagdrupole tandem mass spectrometer(Triple Quad TM 6500⁺). The concentration ranges of plasma from 20. 0 ng/mL to 5 000 ng/mL for amoxicillin and 10. 0 ng/mL to 2 500 ng/mL for clavulanic acid were good linear relationship. The accuracy deviation were ±15. 0% and precision were less than 15. 0% for the intra-assay and inter-assay. The matrix effect and recovery meeted the acceptance criteria, amoxicillin and clavulanic acid were stable under storage and processing conditions. Healthy subjects were given a test preparation of amoxicillin and clavulanate potassium granules 1 bag(125 mg/31. 25 mg/bag)and the reference preparation amoxicillin clavulanate potassium dry mix Suspension “Augmentin^® ” 5 mL(125 mg/31. 25 mg/5 mL)was used to determine the plasma concentration of amoxicillin and clavulanic acid. The Phoenix WinNonlin 6. 4 software was used to estimate the pharmacokinetic parameters of non-compartmental models. The pharmacokinetic parameters of amoxicillin and clavulanic acid were statistically calculated and evaluated the bioequivalence. what′s more, we evaluated the diet on the pharmacokinetics of amoxicillin and clavulanic acid. The analytical method was rapid and sensitive, which was successfully employed in the bioequivalence study of amoxicillin(125 mg/bag)and clavulanate potassium granules(31. 25 mg/bag)for determining the concentration of amoxicillin and clavulanic acid.