A highly sensitive and rapid ultra performance liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous determination of fluticasone propionate (FP) and its major metabolite, fluticasone propionate-17beta-carboxylic acid (FP 17β-CA) in human plasma. The analytes and their deuterated internal standards, FP-d3 and FP 17β-CA-d3 were extracted from 500 μL plasma samples by solid phase extraction on Oasis MAX cartridges. The chromatographic analysis was performed on ACQUITY UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column using methanol-acetonitrile (50:50, v/v) and 2.0 mM ammonium trifluroacetate (ATFA) (85:15, v/v) as the mobile phase. Following separation of the analytes, protonated precursor → product ion transitions (FP: m/z 501.1 → 293.2, FP17β-CA: m/z 453.3 → 293.2, FP-d3: m/z 504.2 → 293.2, FP 17β-CA-d3: m/z 456.3 → 293.2) were monitored on FP 17β-CA a triple quadrupole mass spectrometer, operating in multiple reaction monitoring (MRM) and positive ionization mode. The calibration curves were established in the range of 0.5–100 pg/mL with a correlation coefficient (r2) ≥ 0.9992 for both the analytes. The intra-batch and inter-batch accuracy and precision varied from 95.5-103.4% and 0.74-5.06% across quality controls for both the analytes. The mean assay recoveries for FP and FP 17β-CA were 84.2% and 93.5% respectively. The validated method was successfully applied to support a bioequivalence study of 200 μg FP, administered using nasal spray formulation in 18 healthy Indian subjects. Reproducibility of the method was assessed by reanalysis of 98 incurred study samples.