A quantitative assay based on high-performance liquid chromatography analysis of bile salts and bacterial protein determination was established for investigating bile salt hydrolase (BSH) activity in bacteria isolated from the small intestine of chickens. Bacteria were isolated using various media and were subsequently grouped according to cell morphology, fermentation profile, and 16S ribosomal DNA sequence. Representative isolates from each bacterial group were assayed for BSH activity. The isolates differed in BSH activity with respect to the state of growth and preculturing with and without taurochenodeoxycholate. The highest levels of BSH activity were found with Enterococcus faecium and Clostridium perfringens.