Diphtheria is a serious infection caused by Corynebacterium diphtheriae that usually attacks the respiratory system. The aim of this study is to produce diphtheria toxin using a modified semi synthetic Linggood medium. Also compare the effect of different doses of gamma radiation (0.5, 2, 5, 10, 15, 25 & 40 KGy) and formaldehyde on detoxifying this toxin. SDS-PAGE, protein content and cytotoxicity of toxoid were evaluated. Some biochemical parameters on serum of immunized mice with diphtheria toxoid were measured. The protein content showed that there was no difference with doses (0.5, 2, 5, 10 KGy) whereas, changed with doses (15, 25 & 40 KGy) when compared with standard toxoid. The gamma irradiated toxoids have a non-toxic effect on Vero cells (ATCC® CCL-81, organism: Cercopithecus, Tissue: Kidney). The levels of Urea and Creatinine in serum of all immunized animals groups showed non-significant change compared to control. Meanwhile, activities of serum aspartate aminotransferase (AST) & alanine aminotransferase (ALT) showed no significant difference in case of toxoids irradiated groups (0.5, 2, 5, 10, 15 KGy) while enzymes significantly elevated in toxoid groups (25, 40 KGy and formaldehyde) compared to control animal group. This study revealed that the modified semi-synthetic medium gave a highly purified diphtheria toxin. On the other hand, gamma irradiation with doses (0.5, 2, 5, and 10 KGy) produces safe and effective diphtheria toxoid. It was concluded that this study might be a promising protocol for vaccine production.