How to assess antioxidant activity? Advances, limitations, and applications of in vitro, in vivo, and ex vivo approaches

Abstract Antioxidants are crucial in combating oxidative stress-a key factor in chronic diseases like cancer and neurodegeneration-while also playing vital roles in food preservation, functional food, and nutraceutical development. Their efficacy is assessed through in vitro (e.g., DPPH, FRAP), in situ, ex vivo, and in vivo models, as well as animal and human clinical trials, each offering unique advantages but facing challenges in standardization and physiological relevance. Advances in microfluidics, AI, and nanotechnology are enabling high-throughput screening, while omics integration provides deeper mechanistic insights. This review explores current methodologies, applications in food and pharmaceuticals, and emerging trends, emphasizing the need for rigorous clinical validation and personalized approaches to bridge the gap between laboratory research and real-world antioxidant therapies. Graphical Abstract