A method for measuring 4'-O-tetrahydropyranyladriamycin (THP) and its metabolites in biological samples are described. By reversed-phase high performance liquid chromatography using fluorescence detection, THP and its metabolites were all separated on a single chromatogram within 18 minutes. A linear calibration curve was obtained up to 2,000 ng/ml of THP in plasma. The recovery of THP in the analysis was more than 95% above 5 ng/ml and 87.1% even at 1.25 ng/ml. Thus the lower limit was 1.25 ng/ml in biological samples. Blood levels and urinary excretion in mice and dogs were satisfactory measured by this analytical method.