Abbreviations RIA Radioimmunoassay PSA Prostate specific antigen PCa Prostate cancer PEG Polyethelene glycol NRS Normal rabbit serum Background: Prostate specific antigen (PSA), a glycoprotein secreted exclusively from epithelial cell lining the prostate gland. The level of PSA in human serum may play a critical role in diagnosis of prostatic diseases. Objectives: This study was carried out to set up Radioimmunoassay system for PSA estimation in human serum as a diagnostic tool for Prostatic diseases. Methods: locally produced highly purified PSA was used for preparation of radioimmunoassay components; PSA Polyclonal antibody, radioiodinated PSA Tracer and PSA standards. Optimization and Formulation have been achieved, then validity of this technique confirmed by measuring the quality control parameters; Sensitivity, accuracy, crossreactivity, intra and inter assay precision and method comparison. Results: we found that; Optimized and Characterized Radioimmunoassay system is the most Sensitive, specific, precise and accurate tool for estimation of prostate specific antigen level in human serum. Conclusion: Estimation of PSA with locally produced radioimmunoassay system can be used as a diagnostic tool for diagnosis of prostatic diseases.