MS/MS spectrum matching currently serves as a favored means to identify the concerned metabolites attributing to the accessibility of several famous databases. However, the rule that takes the entire structure into account frequently leads to “0 hit” when inquiring MS/MS (usually MS) spectrum in the databases. Conjugation plays an important role for the high-level structural diversity of metabolites in all organisms, and a given conjugate usually consists of two or more substructures. If MS spectra participate in database retrieval, the structural annotation potential of those databases should be dramatically expanded via identifying substructures. Attributing to the ubiquitous distribution pattern, flavonoid glycosides were deployed as the representative family to justify whether the primary fragment ion termed as Y, resulted from neutral loss of glycosyl residue(s), generated identical MS spectrum with MS spectrum of the aglycone cation namely [A+H]. Because of owning unique ability to measure MS/MS spectrum with the exactly desired exciting energy, linear ion trap chamber of Qtrap-MS was responsible for generating the desired MS and MS spectra. When taking both m/z and ion intensity features into consideration, the findings included: 1) glycosides sharing identical aglycones produced the same MS spectra for Y; 2) different MS spectra for Y occurred amongst glycosides bearing distinct, even isomeric, aglycones; 3) isomeric aglycones generated different MS spectra; and 4) MS spectra for Y agreed with MS spectra of [A+H] when comparing paired glycoside and aglycone. Together, fingerprint comparison between MS and MS spectra could structurally annotate the substructures and further advance MS/MS spectrum matching towards the identification of, but not limited to, aglycones for flavonoid glycosides.