Anthocyanins are water-soluble plant pigments, responsible for the red, purple, and blue colors of many fruits, vegetables, cereal grains, and flowers. They are of interest to nutritionists because of their postulated health benefits, as well as to food processors because of their colorful character as a natural alternative to the use of synthetic dyes. Research concerning their health benefits has become prominent since the late 1990s. Up to now, numerous studies have suggested that anthocyanins are protective against many chronic diseases such as dietary antioxidant [1,2], antimutagenic [3,4], and chemopreventive [5–8] nutraceuticals that contribute to reduced incidences of chronic diseases and the prevention of obesity and diabetes [9,10]. These topics are discussed in detail in the following chapters of this book. Research on the metabolic fate and bioavailability of anthocyanins in vivo become crucial for elucidating pharmacokinetics as well as the possible mechanisms of bioactivity. Therefore, accurate and suitable identification and quantification of anthocyanins and their metabolites must be established. This chapter will present an overview of the current knowledge of how anthocyanin chemistry affects their stability and the chemistry of their metabolites, as well as their bioavailability. More specifically, we will discuss some of the challenges of evaluating anthocyanins in biological samples as well as the different methodologies available for the analysis of anthocyanins and their metabolites in biological samples, including blood, urine, and different tissues or fluids.