Abstract: To study the fragmentation pathways and patterns of asiaticoside and madecassoside by electrospray ionization mass spectrometry (ESI-MSⁿ), the two compounds were analyzed by HPLC-ESI-MSⁿ in positive ion mode and negative ion mode respectively, and multi-stage mass spectrum diagrams of the above two compounds were obtained. Mass spectrometry fragments of asiaticoside and madecassoside were analyzed under positive ion mode and negative ion mode, the stable configuration of sugar ions having the lowest energy was determined by calculation with density functional theory (DFT), and the fragments from the above two compounds mainly were the fragments of the anthocyanin glycosylation after the Glycoside bond of the molecular breaked, and dehydration and cracking rings occured in the fragments of the anthocyanin glycosylation in which the intramolecular hydrogen bonds were not destroyed. The intramolecular hydrogen bonding and dehydrogenation position in the anthocyanin glycosylation were predicted by calculating the molecular space bond length by Monitor Hbonds in the soft Discovery Studio 4.0, from which the positive and negative ions inferred are accord with the anthocyanin glycosylation fragments in the experiment, which confirms the rationality of the law of dehydration and cracking rings in the anthocyanin glycosylation fragments were guided by the law of dehydration and cracking rings of the anthocyanin glycosylation. The fragmentation pathways of the anthocyanin glycosylation fragments are explained by the calculation of the quantum chemistry at a molecular level, and can provide the basis for the chemical structure identification of ursane saponins.