In the present pilot study, for investigating the physiological effects of different types of nondigestible oligosaccharides, we have validated the application of methodologies for the analysis of bile acids and neutral steroids in feces of human subjects. The accuracy of the extraction and chromatographic procedures for the analyses of bile acids and neutral steroids was determined by recovery of added compounds to fecal homogenate. The precision of the above procedures was checked by analyzing these compounds in samples (n = 5) of the same fecal homogenate. Recoveries of added bile acids ranged from 86 and 96%, and those of neutral steroids varied from 81 to 97%. The precision expressed as coefficients of variation of bile acids and neutral steroids ranged from 2.3 to 8.3% and from 6.3 to 11.8%, respectively. The intra- and interindividual variabilities expressed as coefficients of variation of bile acids varied from 1 to 58 and from 0 to 74%, respectively. The same variabilities for neutral steroids ranged from 0.5 to 107% and from 1 to 168%, respectively. The methods validated in the present pilot study were adequate for applying to our forthcoming European Union coordinated major study on the physiological effects of different types of nondigestible oligosaccharides and involving large numbers of samples.