Flavonoids are structurally diverse and among the most ubiquitous groups of dietary polyphenols distributed in various fruits and vegetables. Many have been proposed to be bioactive compounds in the diet that are responsible for lowering the risk of cancer and have been used in chemoprevention studies using animal models of this disease. As for any xenobiotic, to evaluate the potential risks and benefits of bioflavonoids to human health, an understanding of the physiological behavior of these compounds following oral ingestion is needed as well as their absorption, distribution, metabolism, and excretion (ADME). The study on metabolism and bioavailability is very important in defining the pharmacological and toxicological profile of these compounds. Due to great structural diversity among flavonoids, these profiles differ greatly from one compound to another, so that the most abundant polyphenols in our diet are not necessarily the ones that reach target tissues. Therefore, careful analysis of flavonoids and their metabolites in biological systems is critical. Mass spectrometry in various combinations with chromatographic methods has been a mainstay in applications that involve profiling and quantification of metabolites in complex biological samples. Because of its speed, sensitivity and specificity, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the technology of choice for sample analysis. This review describes the chemistry of polyphenols and flavonoids, their ADME, and the various mass spectrometry-based strategies used in the analysis of flavonoids, including future trends in this field. © 2007 American Chemical Society.