In recent years, bile acids (BAs) have received much attention as signaling molecules as well as biosurfactants for lipophilic nutrients. To understand exact BA behavior, the precise distribution of BAs in vivo must be determined. However, to date, it has been difficult to know the precise roles of BA due to variations in BA molecules including conjugated forms. Thus, we reconsidered BA extraction methodology and introduced an ultra performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS) technique for BA analysis. Consequently, we established a rapid and reliable method, using UPLC/ESI-MS, for the analysis of BAs in various biological samples including liver, bile, blood, intestinal contents, and feces. This method enables us to determine the BA profile, including conjugation status, in a single 30 min run. This technique will be a useful tool for the investigation of the roles of BA metabolism in physiological and pathological conditions.