Liquid chromatography hybrid ion trap/time-of-flight mass spectrometry possesses both the MS~n ability of ion trap and the excellent resolution of a time-of-flight and has been widely used to identify drug metabolites and determine trace multi-components in natural products. Collision energy, one of the most important factors in acquiring MS~n information, could be set freely in the range of 10%–400%. Herein, notoginsenosides were chosen as model compounds to build a novel methodology for the collision energy optimization. Firstly, the fragmental patterns of the representatives for the authentic standards of protopanaxadiol-type and protopanaxatriol-type notoginsenosides were obtained based on accurate MS~2 and MS~3 measurements via liquid chromatography hybrid ion trap/time-of-flight mass spectrometry. The extracted ion chromatograms of characteristic product ions of notoginsenosides in Panax Notoginseng Extract were produced under a series of collision energies and compared to screen the optimum collision energies values for MS~2 and MS~3. The results demonstrated that the qualitative capability of liquid chromatography hybrid ion trap/time-of-flight mass spectrometry was greatly influenced by collision energies, and 50% of MS~2 collision energy was found to produce the highest collision-induced dissociation efficiency for notoginsenosides. Addtionally, the highest collision-induced dissociation efficiency appeared when the collision energy was set at 75% in the MS~3 stage. Liquid chromatography hybrid ion trap / time-of-flight mass spectrometry possesses both the MS ~ n ability of ion trap and the excellent resolution of a time-of-flight and has been widely used to identify the drug metabolites and determine trace multi-components in Natural products. Collision energy, one of the most important factors in acquiring MS ~ n information, could be set freely in the range of 10% -400%. Herein, notoginsenosides were chosen as model compounds to build a novel methodology for the collision energy optimization. Firstly, the fragmental patterns of the representatives for the authentic standards of protopanaxadiol-type and protopanaxatriol-type notoginsenosides were obtained based on accurate MS ~ 2 and MS ~ 3 measurements via liquid chromatography hybrid ion trap / time-of-flight mass spectrometry . The extracted ion chromatograms of characteristic product ions of notoginsenosides in Panax Notoginseng Extract were produced under a series of collisions energies and compared to scre en the optimum collimation energies values ​​for MS ~ 2 and MS ~ 3. The results demonstrated that qualitative qualities of liquid chromatography hybrid ion trap / time-of-flight mass spectrometry was greatly influenced by collision energies, and 50% of MS ~ 2 collision energy was found to produce the highest collision-induced dissociation efficiency for notoginsenosides. Addtionally, the highest collision-induced dissociation efficiency occurred when the collision energy was set at 75% in the MS ~ 3 stage.