The present study was designed to systematically investigate the ESI-MSn behavior of a complex 3, 7-O-glycosyl flavonol, kaempferol 3-O-α-L-[2,3-di-O-β-D-(6-E-p-coumaroyl)glucopyranosyl]-rhamnopyranosyl-7-O-α-L-rhamnopyranoside(KO) isolated from Epimedium wushanense, and to address the elimination priority among different glycosylation sites and different sugars/substituents. The direct-infusion ESI-MSn experiment of KO was performed on a hybrid LTQ-Orbitrap Velos Pro mass spectrometer in both negative and positive ion modes by three different fragmentation mechanisms(CID, HCD, and PQD). The CID, HCD, and PQD analyses of KO exhibited remarkable discrimination in respect of the scan range, richness, and distribution of product ions through the entire spectra. KO experienced different fragmentation pathways between two ionization modes: the negative mode CID of KO eliminated the glycosyl portions(priority: 7-sugar > 3-substituent and terminal substituents > inner sugar) and produced aglycone product ions at m/z 284.03/285.04; however, abundant sodium-adduct B32 together with subsequent i,jX30 cleavages were found characteristic for the positive mode CID-MSn. The fragmentation pathways by CID for KO were proposed by analyzing the high accuracy ESI-MSn data. Complementary structural information of KO regarding the aglycone and glycosyl portions was obtained by analyzing the ESI-MSn data in both ionization modes. In conclusion, the LTQ-Orbitrap method facilitates highly reliable qualitative analysis of bioactive flavonoids with three alternative fragmentation modes. The present study was designed to systematically investigate the ESI-MS n behavior of a complex 3, 7-O-glycosyl flavonol, kaempferol 3-O-α-L- [2,3- di-O-β-D- Ep-coumaroyl) glucopyranosyl] -rhamnopyranosyl-7-O-α-L-rhamnopyranoside (KO) isolated from Epimedium wushanense, and to address the elimination priority among different glycosylation sites and different sugars / substituents. The direct- infusion ESI-MSn experiment of KO was performed on a hybrid LTQ-Orbitrap Velos Pro mass spectrometer in both negative and positive ion modes by three different fragmentation mechanisms (CID, HCD, and PQD). The CID, HCD, and PQD analyzes of KO listed remarkable discrimination in respect of the scan range, richness, and distribution of product ions through the entire spectra. KO experienced different fragmentation pathways between two ionization modes: the negative mode CID of KO eliminated the glycosyl portions (priority: 7-sugar> 3-substituent and terminal substituents > inner sugar) and produced a glycone product ions at m / z 284.03 / 285.04; however, abundant sodium-adduct B32 together with subsequent i, jX30 cleavages were found characteristic for the positive mode CID-MSn. The fragmentation pathways by CID for KO were proposed by analyzing the high accuracy ESI-MSn data. Complementary structural information of KO regarding the aglycone and glycosyl portions was obtained by analyzing the ESI-MSn data in both ionization modes. In conclusion, the LTQ-Orbitrap method facilitates highly reliable qualitative analysis of bioactive flavonoids with three alternative fragmentation modes.