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In recent years, there has been an increasing interest in diets rich in fruits and vegetables and this is mostly due to their presumed role in the prevention of various degenerative diseases, such as cancer and cardiovascular diseases. This is mainly due to the presence of bioactive compounds, such as polyphenols, carotenoids, among others. Polyphenols are one of the main classes of secondary metabolites derived from plants offering several health benefits resulting in their use as functional foods. Prior to the use of these polyphenols in specific applications, such as food, pharmaceutical, and the cosmetic industries, they need to be extracted from the natural matrices, then analyzed and characterized. The development of an efficient procedure for the extraction, proper analysis, and characterization of phenolic compounds from different sources is a challenging task due to the structural diversity of phenolic compounds, a complex matrix, and their interaction with other cellular components. In this light, this review discusses different methods of extraction, analysis, and the structural characterization of polyphenolic compounds.