Abstract：Objective To develop an HPLC method to analyze the related substances in josamycin propionate. Methods Due to the complex constitutions of macrolides, the response surface method was used to develop the HPLC method of josamycin propionate based on the design of experiments concept. The organic phase content in mobile phase, the concentration of buffer, and column temperature were selected as significant effects in the design, and resolution of the main peak and the nearest peak in front and numbers of peaks were selected as dependent variables. Results The optimal condition of josamycin propionate HPLC system was as follows: the mobile phase was 15mmol ammonium acetate- acetonitrile (34:66); the column temperature was 40℃; the flow rate was 1.0mL/min and the column used was Xtimate C18 (250mm×4.6mm, 5μm). Conclusion Using the developed method, all peaks were separated well from each other, which was better than methods in pharmacopoeias and literatures. Besides, the validation parameters all met the specifications of standards.