A novel method for separation and on-line characterization of flavonoids from Asparagus officinalis by medium-pressure liquid chromatography coupled to electrospray ionization multi-stage mass spectrometry (MPLC-ESI-MSⁿ) was successfully established. The hyphenation between MPLC and ESI-MSⁿ was designed to keep the split ratio exactly in the range from 1:100 to 1:300. The separation procedure was guided by the chromatogram of ion current of MSⁿ and the structures of compounds were characterized by fragments information at the same time. Consequently, it was proved that MPLC coupled with ESI-MSⁿ was an effective method for separation of compounds from multi-component mixtures with high purity and desired amounts and simultaneous elucidation of chemical structures.