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Flavonoids, such as flavonols and anthocyanins, have been increasingly attributed health-promoting properties for several years. Thus, they are increasingly attracting scientific interest in metabolism and bioavailability research. To investigate the bioavailability of these phytochemicals, new and better methods to examine the metabolism and to quantify the metabolites are needed. Therefore, within the framework of this thesis, new methods for the analysis of the phase II metabolites of flavonols and anthocyanins from physiological samples, such as plasma and urine, were developed and validated. In the blood, most of the flavonoid metabolites are protein-bound, which is why plasma proteins such as human serum albumin represent a particular analytical challenge. With the enzymatic plasma proteolysis, a novel approach was chosen to analyze plasma samples and various peptidases were tested as part of the method development. It could be shown that in addition to conventional sample preparation methods such as SPE and protein precipitation, plasma proteolysis using pepsin is also a suitable approach for the analysis of flavonoid metabolites. The repertory of available valid methods for bioavailability and metabolism research of flavonoids, and possibly other phytochemicals, could thus be expanded by the research work presented here.