Objective A novel Active Pharmaceutical Ingredient (API) pituitrin efficacy component content analysis method was explored, and the method is conformed to the requirements of the pharmaceutical production enterprise testing standard. Methods The reverse phase high performance liquid chromatography (RP-HPLC) was performed on a Zorbax Eclipse XDB-C18(250mm×4.6mm, 5 μm) (P/N 993967-902/ 5063-6600) Column, the column temperature was 25℃, the wavelength of detector was set at 220 nm, flow rate was 1.2 mL/min, 50% Acetonitrile- 0.13mol/L sodium dihydrogen phosphate aqueous solution was used as mobile phase for gradient elution. 20 μL sample solution was injected in each perform. Results The content of oxytocin and vasopressin as pituitrin efficacy components were analyzed by this method with advantage of simple and easy operation, good reliability and high precision. Different biological extraction technology process A and B was used on pituitrin injection production, the content of medicinal ingredients in the product: oxytocin and vasopressin is slightly different, but concentration level is different, which process B concentration is higher than that of A. Conclusion In this study, an effective determination the levels of oxytocin and vasopressin in pituitrin API method was established for a pharmaceutical production enterprise, can provide API pituitrin purification process control. Relevant technical information can be provided in the above, which worked on the biological API pituitrin research and development.