HPLC method for determination of medroxyprogesterone acetate and related substance was established. A Luna C18 column (250 mm × 3.0 mm, 5 μm) was used with the mobile phase of tetrahydrofuran-acetonitrile-water (12:23:65), flow rate was 0.9 mL/min, and detection wavelength was at 254 nm. HPLC could isolate medroxyprogesterone acetate and related substance, the adaptation of system was well, and precision was good (RSD ≲ 2.0%). The solution was stable in 24 h, and quantification limit and detection limit was 0.4 and 0.1 ng, resp. The method was sensitive, specific, and could be used for quality control of medroxyprogesterone acetate and related substance.