Calculus bovis sativus (C. bovis sativus, CBS), with the same pharmacodynamics activities as those of natural C. bovis (CB), has been approved as an ideal substitute in China. However, in the latest edition of the Chinese Pharmacopoeia, only cholic acid and bilirubin are used as the markers for quality control. To overcome current limitations and to further improve its quality control method, a rapid and reliable method was herein developed for simultaneous qualitative and quantitative analyses of taurine and twelve bile acids (BAs) in natural CB and CBS by liquid chromatography coupled with tandem mass spectrometry. Taurine and BAs were separated using a Diamonsil C₁₈ column (150 mm × 2.1 mm i.d., 5 μm) with acetonitrile: water containing 10 mM ammonium acetate (pH 3) as mobile phase in a gradient elution program. Mass spectra were obtained in the negative ion mode through multiple reaction monitoring of respective mass transitions. This method had satisfactory separation efficiency, high sensitivity, little potential interference, and short running time. The average recoveries ranged from 93.0% to 107.3%, with relative standard deviations of less than 9.0% for all thirteen analytes of interest. Taurine and characteristic BAs, especially conjugated BAs, may partially reflect the internal quality of CBS and natural CB. Combining the ratios of cholic acid/deoxycholic acid and unconjugated BAs/conjugated BAs with quantitative data of single BA and chromatographic profiles may be able to control the quality of CBS. In conclusion, this method can be used for explicit identification and quality control of CBS and natural CB.