Objective:To establish a RP-HPLC method for determining the plasma concentration of nonoxynol-9 and to apply the method in studying the pharmacokinetics of nonoxynol-9 in healthy female rabbits. Methods: A single dose of nonoxynol-9 (64 mg to each animal) was given to 6 healthy female rabbits (weighing 2.0-2.2 kg) via vagina. The plasma samples were collected and the concentrations of nonoxynol-9 were determined by RP-HPLC method using DiamonsilTM ODS column (200 mm×4.6 mm,5 μm) and mobile phase of methanol-water (955) at a flow rate of 1.0 ml/min. The excitation and emission wavelengths were 226 nm and 300 nm,respectively. The column temperature was 30℃.The pharmacokinetic parameters were calculated. Results: The calibration curve of nonoxynol-9 showed good linearity over the range of 0.05-5.00 μg/ml; both the intra- and inter-day precisions were lower than 10%. The recovery rates at three concentrations (0.10,0.50,and 2.00 μg/ml) were (101.67±8.12)%,(100.84±5.42)%,and (100.29±3.81)%,respectively. The main pharmacokinetic parameters of nonoxynol-9 were as following: t1/2 (3.75±0.87) h,tmax (3.2±0.4) h,Cmax (2.75±0.32) μg/ml,AUC0-12 (12.72±2.14） μg·h·ml-1,AUC0-∞ (15.05±2.84）μg·h·ml-1,and MRT (6.85±1.13) h. Conclusion: The established method is convenient,sensitive and specific,and can be used for determination of plasma nonoxynol-9 concentration and for its pharmacokinetic studies in rabbits.