Background: Cortisol (hydrocortisone) is the most potent glucocorticoid produced by the human adrenal cortex with about 55-69 μmol (20-25 mg) released daily, and the rate of release has a pronounced diurnal rythm. The plasma concentration is highest in the early morning around 06:00 (140-700 nmol/L), while the nadir is about midnight (~250 nmol/L). Cortisol acts through specific intracellular receptors and has effects in numerous physiologic systems, including immune function, glucose-counter regulation, vascular tone, substrate utilization and bone metabolism. For accurate detection of this low concentration of cortisol in human serum, Radioimmunoassay (RIA) is used; which is known as the most recognized sensitive microanalytical technique for the determination of the very low concentrations of awide range of substances of biological and medical interest. No other analytical method can reach the low detection limits of assayed analytes attained by RIA realted techniques.Applicability, simplicity , to operate and economy of RIA have paved the way for the last advancement in medical research during the past five decades. Materials & Methods: Highly purified Hydrocortisone-21-hemisuccinate and Bovine Serum Albumin used to prepare immunogen (cortisol-21-hemisuccinate: BSA), which is immunized into rabbits to produce cortisol polyclonal antibodies, Na 125I (370 MBq/100μl) carrier and reductant free is the radioactive material used to prepare the tracer (Institute of Isotopes Co.,Hungary). All above materials in addition to prepared standards of cortisol used to prepare radioimmunoassay system to assess cortisol level. Objective: This work was carried to assess and development the technical as well as the economic feasibility of establishing local radioimmunoassay for human serum cortisol. Results: immunogen obtained was of 17 cortisol molecule : 1 BSA molecule ,the dilution of the antibody was chosen to be 1/8000 according to the best displacement percent (76.5%) obtained at this dilution from the the 5th bleed of the 1st rabbit in group A and the prepared tracer was of radiochemical yield percent (58.5%), radiochemical purity (96%)and specific activity (120 μCi/μg) . Conclusion: This RIA-technique method for detection of cortisol level in human serum is simple, accurate and can be used as a decisive diagnostic tool for adrenal status