Abstract: OBJECTIVE To establish and validate a simple, fast and sensitive method for the determination of flurbiprofen in human plasma by HPLC-MS/MS, to study pharmacokinetic characteristics and bioequivalence of flurbiprofen gel paste in Chinese healthy subjects. METHODS Chromatographic separation was accomplished on a InertSustain C₁₈ column(3.0 mm×100 mm, 3 μm) column, temperature was set at 40℃, using a binary gradient with mobile phase methanol- acetonitrile(40:60)(A) and water containing 0.1% formic acid(B) at a flow rate of 0.6 mL·min^-1 and sample preparation was by one- step protein precipitation via methanol. The mass instrument was operated in the negative ion mode, and the monitored transition was set at m/z 243.1→199.2 and m/z 248.0→204.3 for flurbiprofen and IS(flurbiprofen-d5), respectively. RESULTS The total run time in this study was 4.5 min and the retention time of flurbiprofen and flurbiprofen-d5 were 2.43, 2.44 min, respectively. The method was developed and validated over the concentration range of 0.5-100 ng·mL^-1 for flurbiprofen. The inter-batch precision was 1.3%-12.5% and accuracy 97.2%-115.2%. The intra-batch precision was 1.9%-11.6% and accuracy 99.5%-107.8%. The extraction recovery ranged from 103.5%-104.9% and the matrix effect was not significant. Flurbiprofen was proved to be stable in solution and human plasma under the different tested conditions. The validated method was successfully applied to a randomized, open-label, 2-period, 4 sequences, crossover bioequivalence study in healthy Chinese subjects, and the results indicated that bioequivalence was achieved for 2 formulations of per 40 mg paste flurbiprofen gel plaster under external conditions, and both treatments were safe and well tolerated by all study subjects. The pharmacokinetic parameters of flurbiprofen for the reference and test formulations under external conditions were as follows:C_max(19.695±13.159) and (18.135±11.772)ng·mL^-1, T_max(19.375±3.807) and (19.575±4.138)h, AUC_0-∞(534.402±272.792) and (506.529±256.746)h·ng·mL^-1. CONCLUSION The validated method can applied to a bioequivalence study of flurbiprofen in human plasma.