Micro-liquid chromatography (microLC) in conjunction with multistage mass spectrometry (MSn) was introduced to study several major heartsease flavonoid glycosides. High-resolution microLC separation was achieved by using a monolithic poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) column under reversed-phase conditions. The MS/MS and MS3 analysis of the flavonoid components of interest provided data about their glycosylation type and position, nature of their aglycones, and the structure/linkage information of their glycan moieties. With our microLC-MSn approach, four flavonol O-glycosides, nine flavone-C-glycosides, and three flavone C,O-glycosides were characterized in heartsease methanol extract. All of these glycoconjugates were found to be the derivatives of six aglycones: apigenin, chrysoeriol, isorhamnetin, kaempferol, luteolin, and quercetin.