Abstract: AIM：To develop an HPLC method for the determination of etofesalamide in serum, and to study the pharmacokinetics of etofesalamide ointment (ETO) in rats. METHODS: A Shimadzu LC-6A high performance liquid chromatograph equipped with a constant temperature oven set at 40℃ was used. The UV absorbance detector was set at 290 nm, and a 250 mm×4.6 mm(L×ID) column packed with C₁₈ with a 10 μm particle size was used. The mobile phase consisted of methanol-water(0.05% H₃PO₄)=63∶37 with a flow rate of 1.1 mL.min^-1. Ketoprofen was used as the internal standard. The above HPLC method was used to determine the level of etofesalamide in rat serum. The main pharmacokinetic parameters were obtained by 3P97 program. RESULTS: A good linearity was obtained from 10～500 ng.mL^-1 of etofesalamide in rat serum with γ=0.9998. The detection limit was 5 ng.mL^-1. The recovery was more than 94%. The relative standard deviation of within-day and between-day in the level of 20,100 and 500 ng.mL^-1 was 5.82%, 2.43%, 1.78% and 7.77%, 4.01%, 1.09%, respectively. The main pharmacokinetic parameters of ETO after a single external application to rat are shown in table 3. There was good correlation between C_max, AUC and doses, with the correlation coefficient (γ) 0.9986 and 0.9999, respectively. CONCLUSION: The established HPLC method was a simple and accurate method for the determination of etofesalamide in serum. The results of the pharmacokinetic study of ETO showed that it exhibited first order kinetic characteristics.