Owing to the importance of antioxidants in the protection of both natural and man-made materials, a large variety of testing methods have been proposed and applied. These include methods based on inhibited autoxidation studies, which are better followed by monitoring the kinetics of oxygen consumption or of the formation of hydroperoxides, the primary oxidation products. Analytical determination of secondary oxidation products (e.g. carbonyl compounds) has also been used. The majority of testing methods, however, do not involve substrate autoxidation. They are based on the competitive bleaching of a probe (e.g. ORAC assay, β-carotene, crocin bleaching assays, and luminol assay), on reaction with a different probe (e.g. spin-trapping and TOSC assay), or they are indirect methods based on the reduction of persistent radicals (e.g. galvinoxyl, DPPH and TEAC assays), or of inorganic oxidizing species (e.g. FRAP, CUPRAC and Folin-Ciocalteu assays). Yet other methods are specific for preventive antioxidants. The relevance, advantages, and limitations of these methods are critically discussed, with respect to their chemistry and the mechanisms of antioxidant activity. A variety of cell-based assays have also been proposed, to investigate the biological activity of antioxidants. Their importance and critical aspects are discussed, along with arguments for the selection of the appropriate testing methods according to the different needs.

Lipid peroxidation (LP) is the most important type of oxidative-radical damage in biological systems, owing to its interplay with ferroptosis and to its role in secondary damage to other biomolecules, such as proteins. The chemistry of LP and its biological consequences are reviewed with focus on the kinetics of the various processes, which helps understand the mechanisms and efficacy of antioxidant strategies. The main types of antioxidants are discussed in terms of structure–activity rationalization, with focus on mechanism and kinetics, as well as on their potential role in modulating ferroptosis. Phenols, pyri(mi)dinols, antioxidants based on heavy chalcogens (Se and Te), diarylamines, ascorbate and others are addressed, along with the latest unconventional antioxidant strategies based on the double-sided role of the superoxide/hydroperoxyl radical system.

Nanomaterials represent one of the most promising frontiers in the research for improved antioxidants. Some nanomaterials, including organic (i.e. melanin, lignin) metal oxides (i.e. cerium oxide) or metal (i.e. gold, platinum) based nanoparticles, exhibit intrinsic redox activity that is often associated with radical trapping and/or with superoxide dismutase-like and catalase-like activities. Redox inactive nanomaterials can be transformed into antioxidants by grafting low molecular weight antioxidants on them. Herein, we propose a classification of nanoantioxidants based on their mechanism of action, and we review the chemical methods used to measure antioxidant activity by providing a rationale of the chemistry behind them.