The term "bioaccessibility" is a key concept to ascertain nutritional efficiency of food and food formula developed with the aim of improving human health. In this review, working definitions of bioavailability, bioaccessibility, and bioactivity are examined, taking into account the complete sequence of events that take place during the digestive transformation of food into material that can be assimilated by the body, the absorption/assimilation into the cells of the intestinal epithelium, the presystemic metabolism, and, lastly, the development of biologic actions. Comparison among in vivo and in vitro techniques to assess bioaccessibility is accomplished, considering the strengths and limitations of each experimental approach, with a complete description of in vitro procedures applied to determine bioaccessibility of carotenoids. Although a great development has been achieved on the in vitro approaches, these are especially intended for initial screening and should be complemented with in vivo studies, which will remain as the criterion standard for bioaccessibility of nutrients and bioactive compounds at specific target populations. Application of bioaccessibility assessment in foods claiming a health benefit because of their nutrients or bioactive compounds content is described. Measurement of bioaccessibility provides valuable information to select the appropriate dosage and source of food matrices to ensure nutritional efficacy of food products. In addition, in vitro bioactivity measurements to support health benefits of bioactive compounds should be accomplished with estimation of their bioaccessibility, to adequately give nutritional significance to health claims.