Labeling techniques of adrenocorticotropic hormone (ACTH) were described in details. Comparative study was constructed between all tracers that prepared using different oxidizing agents included chloramine-T, iodogen, N-bromosuccinimide and lactoperoxidase & urea-H2O2. The radioisotope of 125I was used in radiolabeling as Na125I in liquid phase. All tracers were purified using PD-10 column and the radiochemical purity was evaluated using paper chromatography electrophoreses charts. Specific activities of tracers were calculated in µCi/µg. The shelf life of tracers in days were determined whereas the validity of radioimmunoassay (RIA) kit will required this factor. Immunoreactivity was examined using polyclonal antibody through RIA liquid phase technique. Maximum binding specific percent and binding nonspecific percent were evaluated. Displacement percent was calculated using minimum and maximum binding values. All the results obtained were used in comparative study included all types of tracers. The results show iodogen was preferred as oxidizing agent than chloramine-T and N-bromosuccinimide. On the other hand enzyme lactoperoxidase was filled completely as oxidizing agent. Because the iodogen is insoluble in water , it can be easily remove from the reaction mixture at the end of reaction without using reducing agent.