Abstract: Steroid hormones are a kind of aliphatic hydrocarbon compounds. They are synthesized in central or peripheral nervous system which are considered as potential biomarkers. High sensitivity method was required for steroid hormones detection for their poor responses and low physiological concentrations. Gas chromatography-mass spectrometry (GC/MS) combined with pre-column derivatization is commonly used in detection of compounds such as steroid hormones. In this work, derivation of oximation conditions were studied based on 12 steroid hormones. Oximation-silylation method was applied for the derivatization of dehydroeppiandrosterone (DHEA), dihydrotestosterone (DHT), androsterone (ADL), epiandrosterone (Epian), pregnenolone (Preg), testosterone (T), estrone (E1), androstenedione (ASD), corticosterone (B),cortisone (E), progesterone (P) and allopregnanolone (AP). Twelve endogenous steroid hormones were processed with methoxyamine (MOX) and N-methyl-N-(trimethylsilane) trifluoroacetamide (MSTFA). Oximation-silanization reactions were carried out on ketone group and hydroxyl group separately which could avoid enolate reaction of ketone group. When processed with MOX，characteristic peaks of M-31⁺ and M-15⁺ were observed in the electron bombardment (electron-impact, EI) mass spectra, mainly due to the loss of —OCH₃ and —CH₃ groups. Therefore peaks of M-31⁺ and M-15⁺ could be considered as the identification for ketoximation. At the same time, characteristic peaks of M-73⁺, M-90⁺ due to the loss of —Si(CH₃)₃ or —O—Si(CH₃)₃ groups from M⁺ were observed when steroid hormones were further silylated with MSTFA. Peaks of M-73⁺ and M-90⁺ could be considered as the basis for hydroxysilylation. Preg, DHEA, AP, ADL, Epian, DHT generated single derivatization with good peak shape. For the existence of conjugated groups in P, ASD and T structures, isomer products generated after derivatization. The ion peak shape was poor and noise was high. For B, and E six derivatives were observed in each derivatives after reaction. Then oximation conditions for endogenous steroid hormones were optimized. GC/MS detection method was obtained after optimization at 80 ℃ for 40 min with the molar ratio of ketone group to MOX 1∶20. With selective reaction monitoring mode (SRM), the detection sensitivity level could achieve to 1 μg/L, and the linear range was 1200 μg/L. When processed with oximation and silylation, the response values of steroid hormones were greatly improved. GC/MS detection with oximation silanization is suitable for the detection of steroid hormones without conjugated groups. This study can provide a detection method for the analysis of related hormonal components.