AbstractThis paper describes a method for the direct gas/liquid chromatographic (GC) analysis of 46 glycine‐conjugated bile acids, which differ from one another in the number, position and configuration of the hydroxyl groups at positions C‐2, C‐3, C‐4, C‐6, C‐7 and/or C‐12. Free bile acids were converted quantitatively on a micro scale to ethyl ester‐trimethylsilyl (Et‐TMS) and methyl ester‐dimethylethylsilyl (Me‐DMES) ether derivatives of the corresponding glycine conjugates. The Et‐TMS and Me‐DMES ethers of the glycine conjugates were chromatographed on an aluminum‐clad flexible fused‐silica capillary column coated with a thin film (0.1 μm) of chemically bonded and cross‐linked methylpolysiloxane. Relative retention time (RRT) and methylene unit (MU) values were determined for the 46 compounds and their GC behaviour was discussed. The derivatization procedure and the retention data would be useful for the direct GC identification of unknown glycine‐conjugated bile acid mixtures