Extracts from wool dyed with sawwort (Serratula tinctoria L.) obtained with methanol/formic acid and methanol/hydrochloric acid solutions were examined by high-performance liquid chromatography with UV detection coupled with electrospray ionization tandem mass spectrometry. Chromatograms and mass spectra were registered in the negative ion mode under various orifice voltages and collision energies, which enabled us to observe signals corresponding to [M - H](-) ions and also Y(-) and/or Y(-•) ions, which were further subjected to fragmentation. The results obtained allowed us to define previously unknown constituents of sawwort, which are proposed as specific markers for its identification: chlorogenic acid and its isomers, luteolin-O-glucuronides, eriodictyol-O-glucuronides, and diosmetin-O-glucuronides. Moreover, it was found that during extraction, flavonoid O-glucuronides react with methanol in the presence of hydrochloric acid, forming stable O-methylated derivatives.