<p>Oxidative stress is a condition caused by an overall surplus of reactive oxygen species (ROS) and its link to many chronic diseases, such as cardiovascular disease, diabetes, neurodegenerative diseases, and cancer, has been well established. Antioxidants are essential to the protection of biological systems by acting as scavengers of free radicals and preventing oxidized damage to biomolecules. Thus, the proper evaluation of the antioxidant capacity of a sample is critical for characterizing natural products, foods, and pharmaceutical products. To evaluate antioxidant activity, numerous in vitro screening methods have been developed using a variety of reaction mechanisms, with the most employed being based on hydrogen atom transfer (HAT) and single electron transfer (SET). The most widely used methods are the DPPH^• radical test, the ABTS^•+ decolorization test, the FRAP test; the ORAC test; and the metal chelation or reducing power tests. All of these provide some degree of complementary information on radical scavenging, reduction potential, and inhibition of oxidative reactions. In addition to activity-based assays, a variety of advanced analytical techniques are also utilized for separating, identifying, and quantifying individual antioxidant compounds within complex matrices. Some of these methods include high-performance liquid chromatography (HPLC) with ultraviolet or mass spectrometric detection (MS). Combining antioxidant assays with chromatographic profiles allows full assessment of antioxidant potential and helps establish links between phytochemical composition and biological effects. This review discusses common in vitro methods for screening antioxidant potential, along with methods of separating antioxidants, and highlights recent developments in reliable evaluation of food, medicinal plant, and nutraceutical antioxidants.</p> Graphical Abstract <p></p>