The present study was designed to prepare a specific safe antiserum for Naja nigricollis using γ-irradiated (1.5KGy and3KGy) venoms. Rabbits were used for active immunization using irradiated venoms (1.5 and 3 kGy) as a toxoid, mice were used for determination of LD50 post immunization and the rats were used for neutralization of the cardiotoxic effect of venom. Results of the immunodiffusion test indicated that the sera of rabbits raised against non-irradiated, 1.5- and 3-kGy γ-irradiated venom, had the same results of precipitin bands. A significant inhibition of phospholipase A2 activities was obtained when neutralized with native, γ-irradiated (1.5KGy and3KGy) venoms. On the other hand, preincubation of the venom ½ LD50 (0.154 mg/kg i.p.) with each antiserum (non-irradiated or irradiated venom) at 37°C for 1 h in a ratio (1:4) produced a significant reduction in the values of creatine kinase and creatine kinase isoenzyme-MB. However, significant elevation in aspartate aminotransferase level and no change in lactate dehydrogenase level were observed. So the results of this study indicated that the irradiated venom treatment reduces the cardiotoxic effect of venom in immunized immunization animals for preparing vaccines.