Free radicals are highly reactive and unstable atoms or molecules that contain at least one unpaired electron. They can be generated chemically in different ways such as redox reactions and ionizing radiation. In biological systems, free radicals are produced as a by-product of normal metabolic processes. A number of free radicals and non-radical reactive species, primarily reactive oxygen species (ROS) and reactive nitrogen species (RNS), are found in the human body. Different ROS/RNS have their own chemical properties, generated through different sources, exhibiting a wide range of reactivates. They exert physiological functions and are necessary for maintaining cell homeostasis. However, excess ROS/RNS can damage cellular lipids, proteins, or DNA to impair normal cellular function. Diet-derived antioxidants are expected to play a supplemental role in scavenging ROS/RNS and to diminish oxidative damage in the human body. Subsequently, a number of chemical-based in vitro total antioxidant capacity (TAC) assays have been developed and widely applied to evaluate the TAC of foods, dietary components, and biological samples. These assays differ from each other greatly in terms of reaction mechanisms, oxidant/free radicals and probe species, reaction conditions, quantification methods, and data expression. In recent years, in vitro TAC assays have been criticized for misinterpretation and misuse of the data. Current knowledge suggests that dietary antioxidants may not work in vivo the same way they are evaluated by in vitro TAC assays. Thus, the justification of using in vitro TAC assays to evaluate dietary antioxidants needs to be reconsidered, while at the same time recognizing appropriate utilizations.