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Background: The presence of oxidant agents yields higher levels of free radical reaction products in erythrocyte membrane proteins and serum proteins. The aim of this study is to investigate the oxidative modifications of recombinant human coagulation factor VIII (rHFVIII) by spectrophotometric analysis. Materials and Methods: In this experimental study, rHFVIII was incubated aerobically with vitamin C and ferro ions in metal catalyzed oxidation (MCO) system for 4 to 28 hr. Carbonyl assay was used as an index of protein oxidation. For this purpose, 2,4dinitrophenyl hydrazine (DNPH) was used. Reaction of this reagent with carbonyl groups produces dinitrophenylhydrazone derivatives that their concentration was estimated by spectrophotometry. Results: Carbonyl groups in rHFVIII changed in the presence of vitamin C and ferro ions. Dose-dependent effects of vitamin C showed a decrease in carbonyl groups of rHFVIII whileferro ions increased oxidation and carbonyl group formation in this protein. Conclusion: These findings indicate that changes in carbonyl groups in rHFVIII are related to the generation of reactive oxygen species. Also, antioxidant mechanisms are activated in a dose- and time-dependent manner.