With an estimated one million fatalities per year worldwide, hepatitis B virus (HBV) infection is the tenth greatest reason for mortality and a severe public health issue. For determining acute infections with HBV or carriers, hepatitis B surface antigen (HBsAg) testing is the most widely used method. Many tiny chemicals and proteins found in human body fluids can be measured quantitatively and qualitatively using radioimmunoassay (RIA). It offers an assay method that is less expensive, more convenient, reliable, fast, sensitive, and dependable. The preparation, optimization, assessment, and validation of the HBsAg RIA system were the topics of this work for the application in clinical purposes. Three reagents were prepared for this local liquid phase RIA technique: HBsAg standards, HBsAg iodine radiotracer, and polyclonal anti-HBsAg antibody. Validation of the proposed assays using some of the performance characteristics of the succeeded immunoassay techniques, including method comparison, specificity, sensitivity, precision, and accuracy were carried out. The findings demonstrated the high sensitivity, precision, accuracy, low coefficient of variation, and lack of cross-reactivity of the proposed assay. There was a significant positive correlation with the commercially available kits as revealed by the method comparison. Overall data imply that this HBsAg- RIA approach is appropriate for standard laboratory applications which would be effectively employed as a crucial diagnostic method for HBV infection.