An automated direct assay system using high performance liquid chromatography was developed for the measurement of RU38486 and its three metabolites (RU42698, RU42848, RU42633) in human serum. Serum concentrations of these compounds were measured up to 144 h following single oral administration of 200 (200 mg group, n = 3) or 400 mg (400 mg group, n = 3) of RU38486 to healthy female volunteers. The serum half-lives (200 mg group-400 mg group) of RU38486, RU42698, RU42848 and RU42633 were 31.8-33.1 h, 41.2-39.3 h, 33.9-36.6 h and 29.2-36.6 h, respectively. Our system could quantify them easily and simultaneously, and was considered to be valuable in studies on the relationship between the pharmacokinetics and the clinical effects of RU38486.