We have attempted to establish the analytical condition of 17 ketosteroids (17 KS), i.e., androsterone (An), etiocholanolone (Eti) and dehydroepiandrosterone (DHEA) for identiˆcation of human urine with gas chromatography-mass spectrometry. First, we optimized the condition of acid-hydrolysis of 17 KS conjugates in human urine. Heating at 100°C for 20 min with 3 of HCl gave maximum yields of liberated 17 KS, and excess HCl and heating for a long period caused a degradation of An and DHEA. Subsequently, we attempted to apply the hepta‰uorobutyryl (HFB) derivatization method using an extraction solvent containing HFB-chloride. This method is convenient because 17 KS are derivatized and extracted simultaneously at room temperature. The highest recoveries were obtained when n hexane containing 5 HFB-chloride and 0.1 triethylamine was used as the extraction solvent. Using this method, we successfully detected the 17 KS in mock forensic samples. We therefore suggest that this method is useful for the identiˆcation of human urine in forensic science.