Tc-99m-cefotaxime sodium (Tc-99m-CEF) was developed and standardized under varying conditions of reducing and antioxidant agent concentration, pH, radioactivity dose, and reducing agent type. Labeling studies were performed by changing the selected parameters one by one, and optimum labeling conditions were determined. After observing the conditions for maximum labeling efficiency and stability, lyophilized freeze dry kits were prepared accordingly. Simple method for radiolabeling of CEF with Tc-99m has been developed and standardized. Labeling efficiency of Tc-99m-CEF was assessed by both radio thin-layer chromatography and radio high-performance liquid chromatography and found higher than 90%. The labeled compound was found to be stable in saline and human serum up to 24h. Two different freeze dry kits were developed and evaluated. Based on the data obtained from this study, both products were stable for 6months with high labeling efficiency. The prepared cold kit was found sterile and pyrogen free. The bacterial infection and sterile inflammation imaging capacity of Tc-99m-CEF was evaluated. Based on the in vivo studies, Tc-99m-CEF has higher uptake in infected and inflamed thigh muscle than healthy thigh muscle.