Technical Abstract: The complexity of sample matrices, coexistence of multiple forms of bioactive phytochemicals, and their interaction of with other cellular components pose a significant challenge for optimize extraction and accurate estimation of bioactive phytochemicals in foods and dietary supplements. This article discusses the significance of optimizing extraction procedures for accurate assay of phytochemicals from different matrices using bioactive isoflavones as a model substrate. Consumption of isoflavone enriched foods such as tofu, miso, natto, and whole soybeans has been associated with reducing the risk of osteoporosis, incidences of hot flashes, menopausal symptoms, cardiovascular disease, and certain forms of cancers. These health promoting properties of isoflavones may be due to their estrogenic and/or antioxidant activities. Isoflavones are known to exist in nature as free aglycons or as conjugates with sugars and/or acids. The wide structural diversity and polarities of free and conjugated isoflavones makes optimum extraction and accurate quantification of isoflavones a challenging task. This paper reviews the different variables that influence quantitative extraction of isoflavones from different matrices. Optimizing procedures for the extraction of isoflavones and other bioactive phytochemicals from foods and plants will allow researchers and regulators to accurately quantify bioactive phytochemicals in different plant matrices. This will enable researchers to provide more precise guidelines on dietary intake and safety levels necessary to achieve optimum health.