Intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes and have significant roles in key cellular processes. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization. This technique consists of the combination of native and 8 M urea electrophoresis of heat-treated proteins where IDPs are expected to run into the diagonal of the gel, whereas globular proteins either precipitate upon heat treatment or unfold and run off the diagonal in the second dimension.