A method of LC-QTOF/MS combining with chemical synthesis has been used to determine the structures of three novel bile acids from bear bile powder. Reference substances of tauroursodeoxycholic acid and taurochenodeoxycholic acid were oxidized by pyridinium chlorochromate. The products were analyzed by LC-QTOF/MS. Total 4 products including 3 isomers were predicted and identified according to the PCC oxidation theory and LC-QTOF/MS results. Bear bile powder samples were dissolved by methanol and analyzed by LC-QTOF/MS. Three unknown peaks were found and identified as 2-[[(3beta, 5beta)-3-hydroxy-7, 24-dioxocholan-24-yl]amino]-ethanesulfonic acid, 2-[[(5beta)-3, 7, 24-trioxocholan-24-yl]amino]-ethanesulfonic acid and 2-[[(5beta, 7beta)-7-hydroxy-3, 24-dioxocholan-24-yl]amino]-ethanesulfonic acid, separately, by matching their results with that of oxidation products above.