Searchable MS/MS spectra libraries, constructed using the results of liquid chromatography coupled with electrospray ionization (ESI) tandem mass spectrometry (LC/MS/MS) with data‐dependent acquisition on an ion trap mass spectrometer, are presented with regard to the identification and confirmation of a variety of closely related flavonoids in a set of biological samples. Flavonoids were found to exhibit a maximum amount of structurally specific MS/MS spectra at 45% of normalized collision energy on the instrument used, without wideband activation. These MS/MS spectra were then searched automatically against a 297‐substance MS/MS library that contains many previously acquired spectra of standard flavonoids. The possible applications of this powerful technique to biological samples are also discussed. Daidzein and genistein were identified through the MS/MS spectra library while searching through LC/MS/MS data for plant and microbial extracts. Moreover, these compounds proved completely distinguishable from other flavonoids of closely related structures in the MS/MS spectra library, using the NIST MS search program. The applicability of the library‐searchable spectra at low concentrations was demonstrated by successful identification of daidzein and genistein at 0.05 and 0.5 µg/mL, respectively. Copyright © 2005 John Wiley & Sons, Ltd.