A rapid and stable method consisting of high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was established for the identification and differentiation of common diastereoisomeric ursane-type triterpenoids at the C-3 position. Two characteristic fragment ions, [M–H–H2O–CO2]– and [M–H–H2O–HCOOH]–, exhibited significant stereochemical effects and were utilized to distinguish 3-OH epimers. Based on reference standards, the abundance of the fragment ion [M–H–H2O–HCOOH]– in 3β-OH compounds in the MS3 experiment was dramatically higher compared to [M–H–H2O–CO2]–; however, for 3α-OH compounds, the product ion [M–H–H2O–CO2]– was noted to be higher than [M–H–H2O–HCOOH]–. Energy-resolved mass spectrometric experiments were carried out to support the differentiation of these diastereoisomeric triterpenoids at the C-3 position. Using this method, a total of nine ursane-type triterpenoids from a plant crude extract, including four pairs of epimers at the C-3 position, were identified and distinguished rapidly. Furthermore, offline Fourier transform ion cyclotron resonance tandem mass spectrometry was also performed to assign accurate elemental compositions. Copyright © 2011 John Wiley & Sons, Ltd.