•First LC–MS/MS method for the simultaneous determination of pyrazinamide and its metabolites.•Most sensitive and rapid method in human plasma compared to all existing methods.•Chromatographic separation of the analytes within 4min.•Consistent and quantitative recovery with minimal matrix interference for all three analytes.•Successful clinical study with healthy subjects and incurred sample reanalysis. In the present work the plasma levels of PZA and its two active metabolites, pyrazinoic acid (PA) and 5-hydroxy pyrazinoic acid (5-OH PA) were determined by a sensitive and rapid LC–MS/MS method. The analytes and their labeled internal standards were extracted from 200μL plasma samples by liquid-liquid extraction with methyl tert-butyl ether: diethyl ether (90:10, v/v) under acidic conditions. Their separation was achieved on a Zorbax Eclipse XDB C18 (100×4.6mm, 3.5μm) column using methanol and 0.1% acetic acid (65:35, v/v) as the mobile phase within 4.0min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions, m/z 124.1→81.1,m/z 125.0→80.9 and m/z 141.0→81.0 for PZA, PA and 5-OH PA respectively in the positive ionization mode. All the analytes were baseline resolved with a resolution factor of 3.3 and 6.4 between PZA and its metabolites, PA and 5-OH PA respectively. The calibration curves were linear from 0.100–30.0μg/mL, 0.03–9.00μg/mL and 0.002–0.600μg/mL for PZA, PA and 5-OH PA respectively with r2≥0.9980 for all the analytes. The intra-batch and inter-batch accuracy and precision (% CV) across quality controls varied from 93.5–106.7% and 1.10–4.57 respectively for all the analytes. The mean extraction recovery of PZA, PA and 5-OH PA was 83.7%, 89.2% and 80.8% respectively, which was consistent at higher as well as lower concentration levels. The% change in the stability of analytes under different storage conditions ranged −6.7 to 7.1 for all the analytes. The method was applied to assess the comparative bioavailability of a 500mg PZA test and reference formulation in healthy subjects. The assay reproducibility was also tested by reanalysis of 22 incurred subject samples.