In this study, we developed a sensitive and efficient analytical approach combining a 96-well plate-based protein precipitation strategy with ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-MS/MS) in order to assess the pharmacokinetic (PK) properties of sivelestat and its metabolite XW-IMP-A in samples of plasma from ALI/ARDS patients with SIRS. The samples were separated via gradient elution with a C18 column (Phenomenex Kinetex, C18, 2.6 μm, 100 Å, 50 × 2.1 mm) using 0.1 % formic acid aqueous solution (A) and acetonitrile-methanol (1:1, V:V) (B) as a mobile phase at a 0.6 mL/min flow rate. UPLC-MS/MS spectra were generated in positive ion mode, and multiple reaction monitoring (MRM) was used to detect the following transitions: m/z 435.1 → 360.0 for sivelestat, m/z 469.0 → 394.0 for sivelestat-IS, m/z 351.0 → 276.0 for XW-IMP-A, and m/z 384.9 → 310.0 for XW-IMP-A-IS. This assay was run for 2.5 min in total, and achieved lowest limit of quantitation values of 2.0 ng/mL and 0.5 ng/mL for sivelestat and XW-IMP-A, respectively, while remaining highly linear from 2−500 ng/mL for sivelestat (r2 ≥ 0.9900) and from 0.5−125 ng/mL for XW-IMP-A (r2 ≥ 0.9900). These validated data were consistent with US Food and Drug Administration (FDA) and European Medicines Agency (EMA) acceptance criteria. In addition, this method was successfully applied to the steady-state PK evaluation of ALI/ARDS patients with SIRS.