Currently, there is increasing interest in developing accurate methods for the quantitative analysis of bile acids (BAs) in biological samples. We have developed a sensitive, fast, and reproducible UPLC-MRM-MS method for BA profiling in serum, liver tissue, or cultured cells of different species (human, rat, and mouse). This method, validated according to FDA guidelines, allows the quantification of 12 non-conjugated, 8 glycine-conjugated, and 11 taurine-conjugated BAs, using 5 additional deuterated BAs as internal standards in a single analytical run. The main features of this analytical approach are its high sensitivity, low sample requirements, versatility, and comprehensive capacity to profile a considerable number of BAs in samples of different species, which make it a valuable tool with potential applications in many research areas focusing on BAs, particularly in toxicological studies.