Abstract: OBJECTIVE To establish the method for simultaneous detection indole and skatole in rat feces from different environments by HPLC. METHODS HPLC-FLD method was used, and the contents were determined in fresh feces of rats from normal environment, SPF breeding area and SPF experimentation area, by Hypersil GOLD C₈ (4.6 mm×250 mm, 5 μm), with water and acetonitrile as the mobile phase for gradient elution. Comparative analysis the content of indole and skatole in rat feces from different environments. RESULTS The conditions for simultaneous determination of indole and skatole in feces of rats were established. In different environments, the content of indole and skatole in feces were significantly different. Compared with the common environment of rats (indole was 2.65 μg·g^-1), the content of indole in feces from SPF experimental and breeding area were 26.09, 6.25 μg·g^-1, the difference were significant (all P<0.01). Compared with the common environment of rats (skatole was 1.82 μg·g^-1), the content of skatole in feces from SPF experimental area and breeding area were 0.43, 0 μg·g^-1, the difference were significant (all P<0.01). CONCLUSION This method has good reproducibility, and strong characteristic, and can be used for the determination of indole and skatole in rats feces.