Ionizing radiation (IR) exposure results in oxidative damage causing cytotoxic and genotoxic effects. Double-strand breaks (DSBs) are considered the most significant DNA lesions induced by ionizing radiation. The present study evaluates the radio protective effect of a novel antioxidant cocktail through quantification of DSB in peripheral blood lymphocytes (PBL) in vivo. The study included 16 consecutive patients who were divided into 2 groups, 6 patients received the novel antioxidant cocktail and 10 control patients. Blood samples were drawn from the patients undergoing bone scan, before the injection of the 99mTc MDP tracer and 2 h after the injection. Quantification of the IR damage was done by Immunofluorescence analysis of the phosphorylated histone, γ-H2AX, used to monitor DSB induction and repair in PBL. The radiation effect of the control group was measured by 2 variables, the average DBSs foci per nucleus and the percent of the DSB bearing cells in PBL. The findings showed a significant increase in the DSBs after isotope injection with an average increment of 0.29 ± 0.13 of foci/nucleus and 17.07% ± 7.68 more DSB bearing cells (p < 0.05). The cocktail treated group showed a lower difference average of − 2.79% ± 6.13 DSB bearing cells. A paired t-test revealed a significant difference between the groups (p < 0.005) confirming the cocktail’s protective effect. The novel anti-oxidant treatment decreases the oxidative stress-induced DNA damage and can be considered as a preventative treatment before radiation exposure.