OBJECTIVE To establish an HPLC method for the determination of related substances of compound norethisterone enanthate injection. METHODS A Phenomenex Gemini C₁₈ column（4.6 mm×250 mm，5 μm） was used with water as mobile phase A and acetonitrile as mobile phase B. Gradient elution was used for the determination and the flow rate was 1.0 mL·min^-1. The detection wavelength was 220 nm. The column temperature was maintained at 35 ℃。RESULTS Norethisterone enanthate, estradiol valerate and their related substances were separated well. The calibration curves of the known related substances, namely norethisterone and estradiol, were linear within the concentration ranges of 5.02-45.2 and 0.505-4.55 μg·mL^-1 with correlation coefficients of 1.000 0 (n=5) and 0.9992 (n=5), respectively. The average recoveries of norethisterone and estradiol were 99.4% and 96.2%（n=3）, with RSDs of 0.9% and 3.3%, respectively. The self-reference standard solution of norethisterone enanthate and estradiol valerate were linear within the concentration ranges of 2.42-121 and 0.242-12.1 μg·mL^-1, and the correlation coefficients were both 1.000 0 (n=6). The LODs of norethisterone, estradiol, norethisterone enanthate, and estradiol valerate were 7.3, 2.0, 0.9 and 1.2 ng, respectively. CONCLUSION The method is accurate, sensitive, and specific. The analsis results of four batches of compound norethisterone enanthate injection showed that there is an evident correlationship between the amount of related substances and storage time of the injection.