Background: DNA damage is one of the major consequences of radiation exposure onto the biological systems. A series of compounds including flavanoids were found to render DNA protection against radiation damage. In this study we elucidated the potential of rutin and rutin hydrate to protect plasmid DNA against damage induced by irradiation. Materials and Methods: DPPH and hydroxyl radical scavenging assays were performed to assess the antiradical potential of rutin and rutin hydrate. Absorption measurements were performed to assess binding parameters of rutin and rutin hydrate with calf thymus (CT)-DNA. Plasmid relaxation assay was performed to compare the radio protective potential of rutin and rutin hydrate against gamma irradiation mediated oxidative damage of pET28 plasmid DNA. Results: DPPH^· assay indicated fast reaction kinetics for rutin and rutin hydrate. However antiradical parameter in terms of EC₅₀ suggested better scavenging capacity for rutin hydrate as compared to rutin. Hydroxyl radical scavenging assay further suggested that both the compounds displayed significant reduction in hydroxyl radicals. Absorption binding study with CT-DNA suggested that rutin hydrate has better binding constant value (K_a = 8.257 x 10⁴ M^-1) compared to K_a = 1.834 x 10⁴ M^-1 for rutin. Plasmid relaxation study demonstrated that plasmid DNA remains predominantly in super-coiled form in the presence of both rutin and rutin hydrate after exposure to 100 Gy of g-radiation. Conclusion: The mechanistic studies suggested that binding and scavenging capacity of rutin hydrate and rutin contributes towards DNA radioprotection. This study may be helpful in devising potent radioprotector molecules helpful for the radiotherapy treatment.