Various chronic diseases like cardiovascular, neurological or metabolic illnesses are linked to oxidative imbalance that arises from the presence and effects of free radicals or reactive oxygen species. Antioxidants neutralize the highly reactive oxygen species, thereby diminishing oxidative stress and minimizing cellular damage caused by these free radicals. This chapter analyzes the primary methodologies used for evaluating antioxidant capacity both in vitro and in vivo, outlining their applications and limitations. In vitro assays, such as oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), cupric reducing antioxidant capacity (CUPRAC) and other electrochemical methods, provide controlled, mechanism-based insights into radical scavenging, metal chelation, as well as redox potential. In vivo models, including the superoxide dismutase (SOD) activity, glycine, cysteine, and glutamate (GSH) assay, enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectrometry (LC–MS), and imaging techniques such as electron paramagnetic resonance (EPR) and fluorescent probes, indicate physiological complexity, bioavailability, metabolism, and systemic distribution. This chapter provides a comprehensive assessment of antioxidant evaluation methods, highlighting the need for increased multifaceted approaches integrating the mechanism-based findings in vitro along with the biochemical and systemic interactions in vivo, thereby improving the applicational relevance of antioxidant research.