SynopsisProtein denaturation was investigated to establish anin vitroevaluation method of surfactants in connection with theirin vivoirritation potency to human skin. Eventually a new method with simplicity and high reproducibility was established by using quantitative analysis with gel‐permeation chromatography (GPC). The protein denaturing potency of the commercially available surfactants was measured by using the developed method. Synergistic reduction in protein denaturation was observed in the mixed systems of anionic and amphoteric surfactants. The synergistic reduction was explained in terms of the physico‐chemical properties of the mixed surfactants. A possible mechanism is the remarkable lowering of the total monomer concentration by the formation of hydrophobic complexes between the anionic and amphoteric surfactants.Relation entre les propriétés physico‐chimiques des mélanges de surfactifs et leur potentiel de dénaturation des