A bioanalytical method for the simultaneous quantification of amoxicillin (AMX) and clavulanic acid (CLV) in human plasma using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been successfully developed and validated. The analytes and amoxicillin-d4 as internal standard were extracted from 100 µL plasma by solid phase extraction using Phenomenex Strata-X cartridges. Chromatographic separation was done on UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) column using acetonitrile: 2.0 mM ammonium formate in water (85:15, v/v) as the mobile phase at a flow rate of 0.400 mL/min under isocratic condition. Mass spectrometric detection was by multiple reaction monitoring with an electrospray ionization source in the negative ionization mode. The response of the method was linear in the analytical range of 10.0-10000 (r2 ≥ 0.9997) and 2.5-2500 ng/mL (r2 ≥ 0.9993) for AMX and CLV respectively. Intra- and inter batch accuracy and precision (% CV) were in the range of 96.1-103.2 % and 1.48-5.88 respectively for both the analytes. The mean extraction recovery was 99.6 and 98.4 % for AMX and CLV respectively. Effect of matrix due to endogenous components on the quantitation and the stability of the analytes under different conditions were extensively studied. Dilution reliability and method ruggedness was also evaluated. The method as successfully applied to a bioequivalence study in 12 healthy subjects using 250 mg amoxicillin + 125 mg clavulanic acid fixed dose combination. The assay reproducibility was successfully demonstrated by reanalysis of 63 subject samples.