Abstract: A rapid analysis method of electrospray-quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF-MS/MS) was established to differentiate ginsenoside isomers Rh₂ and compound K (CK). Based on the analysis of MS/MS product ions with different collision energy in both negative and positive ion mode, ginsenoside isomers Rh₂ and CK are distinguished. The results show that the fragmentation pathway of Rh₂ and CK in negative ion mode is similar. The difference is that the fragment ion abundance of Rh₂ is significantly lower than that of CK with the same collision energy. The fragmentation behavior of Rh₂ is different from CK in positive ion mode, specific fragment ions m/z 465 and m/z 203 are produced with collision energy 20, 40 and 60 eV in CK, but Rh₂ produces the similar specific fragment ions only with collision energy 60 eV in MS/MS. The MS/MS fragmentation pathway is particularly useful in the identification of ginsenoside isomers Rh₂ and CK.