Cefoperazone tracer was radioiodinated with ¹²⁵I by replacing the hydrogen atom with iodonium cation I+ which is known as an electrophilic substitution reaction. All the reaction parameters were studied to produce maximum Radiochemical yield (RCY) of ¹²⁵I-Cefoperazone [¹²⁵I-Cefo] by using 3.7 MBq of Na¹²⁵I, 150 µg of cefoperazone as substrate, 100 µg of Chloramine-T as oxidizing agent in water at 60ºC for 20 min, the RCY of 125I-Cefo up to 90.2% The radiochemical yield was determined practically by electrophoresis 0.02M phosphate buffer with pH 7. A theoretical study was performed using Gaussian 09 to calculate the heat of formation of the expected products. The tracer was separated and purified by high-pressure liquid chromatography (HPLC) with phosphate buffer pH 6.8: methanol (3:1 v/v). The biological distribution of the tracer was studied in normal and infected mice with E-coli bacteria and sacrificed after different time's intervals activity in each organ was counted and expressed as a percentage of the injected activity per organ.