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Plant-based dietary components and additives are known to protect cells from deleterious effect of reactive oxygen species (ROS). Proximate, phytochemical and antioxidant potentials of methanolic extract of defatted Holarrhena floribunda (G. Don) leaves were assessed using in vitro systems such as, 1, 1 diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, nitric oxide radical scavenging and inhibition of lipid peroxidation. Total antioxidant activity was measured using phosphomolybdenum method. Total phenol content and the reductive potential of the extract were also evaluated. The results of the proximate analysis revealed that the leaf contained 0.23% moisture, 12.8% ash, 9.62% crude fat, 23.3% crude fibre, 21.17% protein and 32.68% carbohydrate while the phytochemical constituents included alkaloid, saponin, tannin and cardiac glycosides. The leaf extract of H. floribunda (G. Don) exhibited scavenging activity with IC50 of 12.63, 1,377.00 and 244.00 µg/ml for DPPH, hydroxyl radical and nitric oxide radical respectively. The extract conferred 50% protection at the concentration of 73.80 µg/ml on lipid peroxidation induced by FeSO4 in egg yolk. Total antioxidant capacity equivalent of gallic acid and vitamin C were 195.57 and 519.28 μg/mg of extract respectively and total phenol content equivalent of gallic acid was 1427.87 µg/mg. The reductive potential increased with increasing concentration of extract. The results obtained from this study reveal that the extract is rich in antioxidant components with several mechanisms of eliciting antioxidant actions which provide scientific basis for its use in folk medicine.