OBJECTIVE To make better the RP-HPLC method for the determination of roxithromycin(RM) in human serum. METHODS RM and clarithromycin (internal standard) were extracted from alkalinized serum sample (500 microliters) with methylene chloride. After evaporation of the organic layer, the residue was dissolved in 100 microliters of acetonitrile-ammonium phosphate (1:1, pH 6.0) and washed with n-hexane, then 20 microliters was injected onto a column (5 microns, 15 cm x 4.6 mm) of Penomenex luna C18. The mobile phase was acetonitrile-0.05 mol/L phosphoric acid (39:19:42, adjusted to pH 7.2 with ammonia water) pumped at 1.2 ml/min through the column. The variable wavelength UV detector operated at 0.01 aufs and the wavelength was set at 210 nm. RESULTS The retention times for RM and clarithromycin were 4.4 min and 5.0 min, respectively. Standard curve was linear in the concentration range of 0.25 to 32 mg/L. The detection limit in serum was 0.06 mg/L; the average method recovery 97.4%; the inter-day RSD less than 3.0%. CONCLUSIONS This method was found to be simple, rapid, sensitive and accurate for determination of RM in human serum.