Peroxidation is an important process both in chemistry and biology, and peroxyl radicals play a crucial role in various pathological situations involving lipid and protein peroxidation. A few secondary and tertiary peroxyl radicals can be detected directly by Electron Spin Resonance (ESR). However, primary and secondary alkylperoxyl radicals have extremely short lifetimes and their direct observation is impossible in biological samples. DMPO has been used to trap alkylperoxyl radicals generated in biological systems and the characterization of DMPO-alkylperoxyl spin adducts has been claimed by different authors. However, it was then clearly shown that all the assignments made previously to DMPO-OOR adducts were actually due to DMPO-OR adducts. We have investigated the potential of DEPMPO to characterize the formation of alkylperoxyl radicals in biological milieu. Various DEPMPO-OOR (R = Me, primary or secondary alkyl group) spin adducts were unambiguously characterized and the formation of DEPMPO-OOCH3 was clearly established during the reaction of tert-butylhydroperoxide with chloroperoxidase and cytochrome c.