Abstract: The fragmentation pathways of 4 types and 25 species bile acids in Bile Arisaema were investigated using ultra performance liquid chromatography-quadrupole-time of flight mass spectrometer (UHPLC-Q-TOF MS/MS) by electrospray ionization at positive and negative ion mode. The protonated M+H⁺ or deprotonated M—H^- were observed by ESI-MS, from which the molecular weights were deduced. The MS/MS data of M+H⁺ or M—H^- ions provided fragmentation information of the compounds. The main and typical fragmentation pathways of different bile acids were neutral losses of H₂O and CO₂ units in the substituent on the mother nucleus. In particular, the functional groups in the C17 position of steroid mother nucleus were broken and provided characteristic fragmentation information. Meanwhile, the cleavages of A and C ring were also observed at the positive ion mode of the free bile acids. The common fragment ions of m/z 175, 161 and 147 were characteristic ions. The ions of neutral loss were also observed at the negative ion mode of the free bile acids, generating ions of M—H—CO₂^-, M—H—H₂CO₂^- and M—H—H₂O—H₂CO₂^-. Taurine type bile acid could be broken and provided taurine group fragment ion m/z 126.022 6 at the positive ion mode. Glycine type of bile acid could also be broken and provided glycine group fragment ion m/z 158.081 8 at the positive ion mode. Taurine type bile acid could be broken and provided high abundance of SO3- fragment ion m/z 79.956 8, NH₂—CH₂—CH₂—SO₃^- fragment ion m/z 124.007 3 and CH₂=CH₂—SO₃^- fragment ion m/z 106.980 3 at the negative ion mode. Glycine type of bile acid could also be broken and provided high abundance of NH₂—CH₂—COO^- fragment ion m/z 74.042 6 at the negative ion mode. In addition, the characteristic ion of m/z 402.301 3 with ketone group would be formed by the cleavage of single ketone group bile acid at the negative ion mode. It was difficult to form stable ions for bile acid esters at the negative ion mode. It could be broken and provided NH₂—CH₂—COOCH₃⁺ fragment ion m/z 90.055 7. The fragmentation behavior of bile acids was reasonable and could provide the basis for the structures elucidation and identification in the Bile Arisaema. It will provide the basis for rapid analysis and identification of samples which contain bile acids.