For orally administered drugs, the metabolism of a drug by the gut flora plays an important role in the bioavailability, activation and disposition of the drug in vivo. However, no in vitro system is currently available to evaluate the metabolism of a drug by the gut flora before the drug is absorbed into the body. This paper presents an in vitro metabolic system in an anaerobic environment that could be used to evaluate the metabolism of an endogenous compound, cholic acid, and a xenobiotic compound, ginsenoside Rg3. We showed that the proliferation of the anaerobic bacteria of the gut content of hamsters produced a similar composition of gut flora in a culture medium for yeast to that in vivo. Incubation of ginsenoside Rg3 and cholic acid in the anaerobic in vitro system efficiently produced the metabolites Rh2 and deoxycholic acid, respectively, similar to those seen in the gut content in vivo. In comparison with in vivo analysis, this anaerobic in vitro metabolic system is convenient, reproducible, economic and animal saving, and can easily be applied to assess the transformation and disposition of a drug before it enters into the circulatory system.