A gas chromatography (GC) method for the simultaneous determination of Androst-4-ene-3,17-dione (4-AD),3β-methoxymethyl-ether-dehydrepiandrosterone and dehydroepiandrosterone (DHEA) from a culture of microbial fermentation for the production of transformable 3β-methoxymethyl-ether-phytosterols was described.The samples were quantitated by internal standard method,and they were separated by GC with DB-1 capillary column and detected by FID.The column and the injector were operated at 280 ℃,the detector was set as 310 ℃ and the carrier gas was nitrogen,flowing at a rate of 1 mL min-1.The samples were quantitated by internal standard method.Under the conditions,all components in culture were well separated and rectilinear responses were obtained in the range of 0.2 ～ 1.0 g/L (4-AD),0.1 ～ 0.9 g/L (3β-methoxymethyl-ether-dehydrepiandrosterone),and 0.2 ～ 1.0 g/L (DHEA).The correlation coefficients (R2) are 0.996,0.992 and 0.996 respectively.Recoveries of 4-AD,3β-methoxymethyl-ether-dehydrepiandrosterone and DHEA added to culture were (97.88 ± 0.74) ％,(99.39 ± 1.07) ％and (98.91 ± 1.09)％ respectively.The method is accurate,rapid and suitable for routine analysis.