Abstract: OBJECTIVE To establish a reliable method for whole blood collection and bioanalysis method for the bioequivalence study of suplatast tosilate(ST) granules. METHODS Blood was collected into heparin tubes containing NaF/EDTA under ice bath conditions. After processing, the separated plasma was split into 2 aliquots into each heparin tube containing 0.5% formic acid. Plasma sample(20 μL) was pre-treated by protein precipitation, and ST-D5 was used as the internal standard. Chromatographic separation was achieved through a normal phase silica columnAgilent Polaris 3 Si-A column(3.0 mm× 100 mm, 3 μm), the mobile phases was 10 mmol·L^–1 ammonium acetate solution and methanol-acetonitrile(1︰1). The flow rate was 0.80 mL·min^–1, the organic proportion was 50% with isocratic elution. Then with the mass spectrometer operated in positive electrospray ionization mode using multiple reaction monitoring, ST and ST-D5 were quantitative analyzed by detecting the ion transitions of m/z 328.1→266.3, 333.1→271.3. RESULTS The method showed linearity over the concentration range of 1 -200 ng·mL^–1was good(r=0.999 8). The extraction recoveries of ST and ST-D5 were between 105.0% -117.1%. Intra- and inter-day accuracy were in the ranges of 95.67% -108.00%(precision≤9.10%) and 99.10% -105.00%(precision≤7.66%), respectively. Stability of ST in whole blood and plasma were acceptable. CONCLUSION The established method is stable, accurate, and reproducible for ST with small sample volume, and meets the requirements of bioequivalence studies of ST granules.