Endogenous steroid hormones have been reported as an important role to metabolism control, inflammation regulation and development of reproductive organ. These endogenous steroid hormones are related with human disease such as breast, ovary cancer. In this study profiling method of endogenous steroid hormones as trimethylsilyl (TMS) derivative in ovarian cancer tissue was developed by high resolution gas chromatography–tandem mass spectrometry (GC−MS/MS). Characteristic ions in MRM mode with electron ionization (EI) permitted the accurate and selective analysis of endogenous steroid hormones. In optimal conditions, this method showed good linearity (r ≥ 0.9989) with limit of detection (LOD) of 2.5−48.5 pg and limit of quantification (LOQ) of 8.5−161.6 pg. The repeatabilities of intra− and inter−days as a percentage of relative standard deviation (% RSD) varied from 0.2−10.0 (% RSD), respectively accuracy as a percentage relative error (% RE) was from -8.2 to 8.2 (% RE). The recovery ranged from 87.7 ± 6.4 % to 99.7 ± 9.7 %. When applied to tissues of 11 ovarian cancer patients, total eight endogenous steroid hormones of dehydroepiandrosterone, estradiol, testosterone, pregnenolone, estriol, progesterone, cortisone and cortisol were identified and determined by GC−MS/MS. Therefore, the present endogenous steroid hormone profiling method in ovarian tissue by GC−MS/MS will be useful for the monitoring and screening of ovarian cancer in clinical fields.