It is known that n-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid and eicosapentaenoic acid, are rapidly oxidized in vitro. Nε-(propanoyl)lysine (propionyllysine, or PRL) is formed from the reaction of the oxidized products of n-3 PUFAs and lysine. To evaluate the oxidized n-3 PUFA-derived protein modifications in vivo, we have developed detection methods using a novel monoclonal antibody against PRL as well as liquid chromatography–mass spectrometry (LC/MS/MS). The antibody obtained specifically recognized PRL. A strong positive staining in atherosclerotic lesions of hypercholesterolemic rabbits was observed. We have also simultaneously identified and quantified both urinary PRL and urinary Nε-(hexanoyl)lysine, using LC/MS/MS using isotope dilution methods. The level of urinary PRL (21.6±10.6 μmol/mol of creatinine) significantly correlated with the other oxidative stress markers, 8-oxo-deoxyguanosine, dityrosine, and isoprostanes. The increase in the excretion of amide adducts into the urine of diabetic patients was also confirmed compared to healthy subjects. These results suggest that PRL may be good marker for n-3 PUFA-derived oxidative stress in vivo.