4-Hydroxynonenal (HNE) is a product of lipid peroxidation. It has been often used as a biomarker of endogenous lipid peroxidation and its concentration is increased in several diseases. But HNE is not only formed during lipid peroxidation occurring in the body. Some authors have shown that it is also present in oxidized oils and in meats. The aim of this study is to compare the effect of food composition (heme iron, fatty acid composition) or freeze-drying on HNE formation in foodstuffs. The methodology is based on extraction/purification procedure followed by HPLC separation with UV detection. As HNE is chemically very reactive and binds easily to proteins, we used radiolabeled HNE to calculate extraction efficiency, so total HNE can be estimated as only free HNE can be measured. The concomitant presence of both heme iron and omega 6 fatty acids, such as linoleic acid, is important for HNE formation in foodstuffs. Freeze-drying increases this formation.