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Purpose To explain the observed radio-protection properties of an azo compound, 2-(2-hydroxyphenylazo)-indole-3∕-acetic acid (HPIA). Materials and methods Mechanism of radioprotection by HPIA was attempted using the stable free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) using UV-Vis and electron paramagnetic resonance (EPR) spectroscopy. The radical destroying ability of HPIA was studied by depletion of reactive oxygen species (ROS) in WI 38 lung fibroblast cells. Results & Discussion Studies indicate HPIA interacts with radical intermediates formed in solution following irradiation by 60Co γ-rays. As a result, reactive radical intermediates do not cause any damage on chosen substrates like thymine or calf thymus DNA when irradiated in presence of HPIA. The study showed that reactive intermediates not only react with HPIA but that the kinetics of their reaction is definitely faster than their interaction either with thymine or with DNA. Had this not been the case, much more damage would have been observed on chosen substrates following irradiation with 60Co γ-rays, in the presence of HPIA than actually observed in experiments, particularly those that were performed in a relatively high dose. Experiments reveal radiation induced-damage caused to thymine in presence of HPIA was ~ 136 to ~ 132times that caused in its absence under different conditions indicating the radio-protection properties of HPIA. In case of calf thymus DNA, damage in presence of HPIA was much lower than in its absence. A fluorometric microplate assay for depletion of ROS by detecting the oxidation of 2′,7′-dichlorofluorescin-diacetate (DCF-DA) into the highly fluorescent compound 2′,7′ dichlorofluorescein (DCF) indicated HPIA brought about a considerable check on ROS-mediated damage to cells by scavenging them right away. Conclusion The study indicates HPIA may be an antioxidant supplement during radiotherapy., Pharmaceutical chemistry, 2-(2-hydroxyphenylazo)-indole-3ï,¤-acetic acid (HPIA), Thymine, Calf thymus DNA, DPPH, DCFDA, Radioprotection