Antioxidants are pivotal in mitigating oxidative stress by neutralizing reactive oxygen species (ROS), thus reducing cellular damage and potentially preventing chronic diseases. Although in vitro assays are preliminary tools for assessing antioxidant activity, they are limited in replicating living organisms’ complex pharmacokinetic and pharmacodynamic profiles. This chapter emphasizes the importance of in vivo testing for accurately evaluating antioxidant compounds’ bioavailability, metabolism, and physiological relevance. In vivo models allow for a comprehensive analysis of pharmacokinetic parameters, including absorption, distribution, metabolism, and excretion (ADME), which are crucial in determining therapeutic efficacy. Moreover, in vivo studies reveal the influence of factors such as dose–response relationships, tissue specificity, and interindividual variability, which are often overlooked under in vitro settings. The application of antioxidants extends across fields such as pharmaceuticals, nutraceuticals, and preventive medicine, where their roles in reducing inflammation, slowing aging processes, and potentially lowering the risk of conditions like cardiovascular diseases and neurodegenerative disorders are actively explored. Incorporating in vivo studies yields crucial insights into antioxidants’ practical efficacy and safety profiles, facilitating the development of clinically relevant formulations and optimized therapeutic strategies. The comparative analysis supports a translational approach that connects in vitro and in vivo findings, advancing the development of effective antioxidant interventions for disease prevention and health optimization.