Polyphenols belonging to the class of secondary metabolites of plants and microorganisms play an important role as bioactive food constituents as well as contaminants. Structure elucidation of polyphenols in plant extracts or polyphenol metabolites, especially those arising during biotransformation, still represents a challenge for analytical chemistry. Various approaches have been proposed to utilize fragmentation reactions in connection with mass spectrometry (MS) for structural considerations on polyphenolic targets. We compiled and applied specific liquid chromatography (LC)–electrospray ionization in positive mode [ESI(+)]–tandem MS (MS/MS) and gas chromatography (GC)–(electron impact, EI)–MS/MS fragmentation reactions with a special focus on the analysis of isoflavones, whereby this technique was also found to be extendable to determine further polyphenols. For ESI(+)-MS the basic retro-Diels–Alder (rDA) fragmentation offers information about the substitution pattern in the A- and B-rings of flavonoids and the elimination of a protonated 4-methylenecyclohexa-2,5-dienone (m/z = 107) fragment can be used as a diagnostic tool for many isoflavanones. For GC-(EI)-MS/MS analysis after derivatization of the analytes to their trimethylsilyl ethers, the elimination of methyl radicals, tetramethylsilane groups or the combined loss of two methyl groups can be shown to be specific for certain substitution patterns in polyphenols. The applicability of the fragmentation reactions presented is demonstrated exemplarily for three derivatives of the isoflavone irilone. With the help of these fragmentation reactions of the two MS techniques combined, a reliable identification of polyphenols is possible. Especially in such cases where NMR cannot be utilized owing to low analyte amounts being available or prior to purification, valuable information can be obtained.