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Oxidized low density lipoprotein (Ox-LDL) is implicated in a variety of oxidative diseases. To clarify the mechanisms involved and facilitate the investigation of therapeutics, we previously developed a detection method for lipid-derived radicals using the fluorescent probe 2,2,6-trimethyl-6-pentyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)piperidine-1-oxyl (NBD-Pen). In this study, NBD-Pen was used to detect lipid-derived radicals in Ox-LDL from in vitro and in vivo samples using an iron overloaded mouse model. By following the timeline of lipid radical generation using this method, the iron overloaded mice could be successfully treated with the antioxidant Trolox, resulting in successful lowering of the plasma lipid peroxidation, aspartate transaminase and alanine transaminase levels. Furthermore, using a combination therapy of the chelating agent deferoxamine (DFX) and Trolox, liver injury and oxidative stress markers were also reduced in iron overloaded mice. The NBD-Pen method is highly sensitive as well as selective and is suitable for targeting minimally modified LDL compared with other existing methods.Oxidized LDL is involved in pathogenesis of various diseases.Lipid radicals are key molecules in LDL oxidation pathway.High selective fluorescent detection probe for lipid radical in LDL was used.Trolox treatment based on lipid radicals’inhibition was effective.Combination with chelating agent improved therapeutic effect of trolox.