Snake bile, a precious traditional Chinese medicine (TCM), was used as the major ingredient of some Chinese patent drugs, such as Shedan Chuanbei powder and Shedan Chenpi powder for hundred years. However, there is still requirement for the comprehensive and definite composition of bile acids in snake bile. In order to rapidly identify the bile acids constituents in snake bile to avoid the adulteration, ultrahigh-performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/ESI-QTOF-MS/MS) has been applied to conduct a qualitative analysis on snake bile acids. ESI ion source was used for mass spectra, and data were collected in both positive and negative ion mode. 16 kinds of reference standards, attributed to free bile acids, taurine- and glycine- conjugated bile acids, were detected and their MS behaviors were summarized. In negative ion mode, the diagnostic ions of free bile acids were obtained via the neutral losses of H₂O and CO₂ molecules; the diagnostic ions of taurine-conjugated bile acids were at m/z 124.0068 ([C₂H₆NO₃S]^-), m/z 106.9803 ([C₂H₃O₃S]^-) and m/z 79.9568 ([SO₃]^-); the diagnostic ion of glycine-conjugated bile acids was at m/z 74.0242 ([C₂H₄NO₂]^-). In positive ion mode, dehydration ions, amide bond cleavage ions, and reversed Diels-Alder at A-ring ions were detected in every kind of reference. These reference MS behaviors were used for identifying bile acids without reference standards in snake bile. As a result, totally 15 compounds, including 4 pairs of isomers, were identified by comparing the retention time, exact molecular mass and fragmentation behaviors with reference standards, respectively. Tauro-3β,7α,12α-trihydroxy-5β-cholenoic acid, Tauro-△⁸-3β,7α,12α- trihydroxy-5β-cholenoic acid, Tauro-3α,7α,12α,23R-tetrahydroxy-5β-cholenoic acid, and Tauro-3α,7α-dihydroxy-12-oxo-5β-cholenoic acid, Taurocholic acid, Glycocholic acid, Taurochenodeoxycholic acid, Taurodeoxycholic acid and Cholic acid were detected as the common bile acids in snake bile. Tauro-△⁸-3β,7α,12α-trihydroxy-5β-cholenoic acid, Tauro-3α,7α,9α,16α-tetrahydroxy-5β-cholenoic acid, Tauro-3α,12α,17R,22R-tetrahydroxy-5β-cholenoic acid, and Tauro-△^1,8-3β,7β,12α-trihydroxy-5β-cholenoic acid were firstly reported in this study.