Flavonoids are a major class of plant phenolics that are widely distributed in the human diet and have been related to health promotion. They may occur in their natural sources in free forms (aglycones), as glycosylated or acylated derivatives, or as oligomeric and polymerized structures. This structural diversity affects their physicochemical behaviour and complicates their analysis. Thus, there is not a single standardized procedure that can be recommended for all flavonoid groups and/or type of samples, and the procedures have to be optimized depending on the nature of the sample and the target analytes. Furthermore, when dealing with the analysis of flavonoids biological samples (i.e., human and animal fluids and tissues) some differential aspects have to be taken into account; the nature of the compounds that can be found in those samples may differ from that present in plants and food, and flavonoids and metabolites occur in much lower concentrations, which make their analysis still more challenging. In this review the main techniques for extraction and analysis of flavonoids in foodstuffs and biological fluids are revised, as well as their occurrence in foods and beverages and available databases.