Immunodiagnostic tests based on in vitro enzymatic methods are mostly preferred to in vivo methods for the detection of cellular immunity due to their cost, applicability, repeatability, and higher sensitivity. Therefore, the development of a quantitative sandwich ELISA prototype detecting bovine IFNg (bIFNg) was planned. In this study, two clone originated monoclonal antibodies were used for capturing the plates, and biotin conjugated monoclonal Ab was used as a secondary antibody in sandwich ELISA. Different approaches such as inter-and intraassay precision and dilution parallelism were evaluated for test validation. The correlation of coefficient variation that is over 95% and 5% variations in precision detection, with an estimated concentration of 98.7%, makes this prototype an alternative test for detection and quantitation of bIFNg. Trials intending to detect the shelf-life of the prototype for 6 months demonstrated that the detection limit was found to be approximately 100 pg/mL of the capturing Ab from clone I with a detection range of 10% CV throughout that period. Preliminary results show that the prototype can be applied to in vitro bovine tuberculosis detection for plasma samples at least for the validation period of 6 months.