The main objective of the present study was the preparation of the primary reagents of cortisol radioimmunoassay (RIA) using liquid phase double antibody technique. Three basic components were prepared and characterized to obtain valid and accurate system. These components were polyclonal anti-cortisol antibody, 125Icortisol RIA tracer and cortisol standards. Cortisol requires conjugation step to be real antigen. Therefore, in this study this mandatory conjugation step was performed & characterized. Then formulation, optimization and validation of this liquid phase RIA technique were carried out. This assay is precise, specific and sensitive enough for using as a diagnostic tool for the adrenal cortex status