AbstractPerforming 2‐DE of lipid‐rich multilamellar membranes like myelin is a cumbersome task. However, for understanding its molecular organization and changes during diseases, identification of proteins of myelin is essential. Although the 2‐D‐proteomic approach of myelin has been employed to understand the myelin proteome, representation of myelin proteins in its entirety is still a challenge. 2‐DE profiling of myelin proteins is very important for the detection of immuno‐reactivity to myelin proteins from various biological fluids following Western blotting in diseases like multiple sclerosis. Here we developed a novel approach by exploiting the thermodynamic principles behind detergent‐mediated solubilization of myelin membranes without any conventional processing of myelin involving precipitation of myelin proteins. We show that the addition of myelin to ASB‐14‐4 resulted in significant increase in protein representation of myelin in 2‐DE compared with the addition of ASB‐14‐4 to myelin. Moreover, the number and resolution of spots are significantly higher in myelin to ASB‐14‐4 strategy than other strategies of myelin sample processing such as ASB‐14‐4 to myelin or ethanol or acetone or methanol–ammonium acetate precipitation of myelin proteins. In addition, the step involves no precipitation that selective removal of any proteins as a result of precipitation is nil and a qualitative representation of myelin proteins in a 2‐D gel is achieved.