Abstract: AimTo quantify progesterone(P) and one of its metabolites 20α-hydroxy-4-pregnen-3-one (20α-OHP) in rat plasma and uterus after im administration of progesterone. MethodsPlasma and uterus samples were prepared by liquid-liquid extraction and separated through Shimadzu VP-ODS column (150 mm×4.6 mm ID, 5 μm). The mobile phase consisted of acetonitrile and water (60∶40, adjusted to pH 4.0 with phosphoric acid). The detector was set at 240 nm. Norgestrel was used as the internal standard. ResultsC_max of P in plasma was (508±62) μg·L^-1, T_max was (3.2±0.4) h, T_1/2(k_e) was (10±4) h and mean AUC_0-48h was (5 886±1 573) μg·L^-1·h. The maximum concentration of P in uterus was(1.7±1.1) μg·g^-1 and the peak time was (5.2±1.1) h. 20α-OHP showed a similar T_max with P. ConclusionThe method is accurate and convenient. It can be used to determine P and its main metabolite 20α-OHP simultaneously for studying their preclinical pharmacokinetics.