Flavonoids receive considerable attention in the literature, specifically because of their biological and physiological importance. This review focuses on separation and detection methods for flavonoids and their application to plants, food, drinks and biological fluids. The topics that will be discussed are sample treatment, column liquid chromatography (LC), but also methods such as gas chromatography (GC), capillary electrophoresis (CE) and thin-layer chromatography (TLC), various detection methods and structural characterization. Because of the increasing interest in structure elucidation of flavonoids, special attention will be devoted to the use of tandem-mass spectrometric (MS/MS) techniques for the characterization of several important sub-classes, and to the potential of combined diode-array UV (DAD UV), tandem-MS and nuclear magnetic resonance (NMR) detection for unambiguous identification. Emphasis will be on recent developments and trends.