We have attempted to establish the analytical condition of 17-ketosteroids (17-KS), i.e., androsterone (An), etiocholanolone (Eti) and dehydroepiandrosterone (DHEA) for identification 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 heptafluorobutyryl (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 identification of human urine in forensic science.