Flavonoids continue to attract wide attention as possible very useful agents for combating free radical pathologies, i.e. the pathological states associated with free radical overproduction. Commonly used methods for the analysis of plant flavonoids include high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). On the other hand, the soft-ionization approach based on electrospray ionization (ESI-MS) permits highly selective analysis of complex matrices. In this work, we examined firstly the ESI-MS behaviour of representative aglycones and glycosides of flavonols, flavones and isoflavones with the aim of suggesting a possible relationship between structure and mass spectra. Using HPLC coupled to a diode array detector (DAD) for on-line UV spectra acquisition, and in parallel to ESI-MS for mass spectra (LC/DAD-ESI-MS), we have developed methodology to observe flavonols directly in tomato puree extract. In this way, it has been possible to detect intact flavonol glycosides in tomato extracts and to characterize a flavonol trisaccharide. For the first time, using LC/ESI-MS, it has been possible to detect intact flavonol glycosides in plasma of healthy volunteers and to provide further evidence on the absorption of flavonoid glycosides after consumption of common vegetables like tomatoes.