In this paper, we report the micro concentration of Ru(III) used as homogenous catalysis in the oxidation of levothyroxine by N-bromosuccinimide and the results of a spectrophotometric investigation of the mechanistic pathway. Such studies are helpful in gaining an insight into the interaction of metal ions through the investigation of the mechanistic pathway of levothyroxine in redox reaction.The reaction stoichiometry is 1:1 in the presence of catalyst. The oxidation products of levothyroxine have been identified as 2-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]acetaldehyde and succinimide by TLC, UV and FTIR. The reaction rate was examined with reference to variations in ionic strength, solvent polarity, free radicals, halides ions, mercuric acetate for the sequestration of Br− in the reaction mixture and the addition of the product succinimide (RNH). The reaction constants involved in the mechanism were computed. The overall activation parameters have been evaluated from the Arrhenius plot. The observed results have been explored by proposed mechanisms, and the relative rate laws have been evaluated further.