In order to study functionality and pharmacodynamics of any important biological molecule such hormones and enzymes, it takes alot of time and complexed stratigies to do that. Here we study the incidence of new recombinant human insulin prevalence in body tissues in real time with aid of radiotracing technique. A new designed model of recombinant-human insulin and produced in E.coli suggested to be functional without post translational modification, has been radiolabeled with Technetium-99m, then tracing its biodistribution with 99mTc in mice. The new construct of human recombinant Insulin protein subjected to the formation of 99mTc-complex using sodium dithionite(Na2S2O4) as a reducing agent, whole reaction conditions subject to optimization of the pH, tempature, substrate amount and a reducing agent amount. Then 99mTc-Insulin complex has been intravenously administrated for biodistribution study invivo in Albino Swiss mice. The optimized conditions for preparing the 99mTc–insulin complex with the highest radiochemical yield(93.3%) disclosed that using 100 μg of insulin in the presence of 20μg of a reducing agent sodium dithionite(Na2S2O4) at pH 8 and within 30 minutes reaction time. The radioactive complex of 99mTc-Insulin gives a much better result after IV injection, where no accumulation in distinect organ. And due to aqueous nature of Insulin, it show clearance from both renal and hepatic route, Also prevalance of Insulin in body according every organs mass or size, have a great impact of functionality of recombinant-insulin molecule. Consequently, this method seems to be much rapid and effective for evaluation of biological molecule invivo via radioactive tracing technique.