Most proteins in nature are in equilibrium with partially and globally unfolded conformations. In contrast, kinetically stable proteins (KSPs) are trapped by an energy barrier in a specific state, unable to transiently sample other conformations. Kinetic stability (KS) appears to be a feature used by nature to allow proteins to maintain their activity under harsh conditions and to safeguard the structure of proteins that are intrinsically prone to misfolding and aggregation. The biological significance and structural basis of KS remains poorly understood due to the lack of a simple method to identify this infrequent property of protein. We showed a correlation between KS and a protein's resistance to the denaturing detergent SDS, and subsequently developed a diagonal 2D (D2D) SDS/PAGE assay to identify KSPs in complex mixtures. We applied this method to the lysate of E. coli and upon proteomics analysis identified 44 non-redundant proteins with known 3D structure. Thus, a protein's resistance to SDS may be used to probe its kinetic stability, and D2D SDS/PAGE will make possible the proteomics-level identification of KSPs in different cells or organisms. Several applications of D2D SDS/PAGE will be presented to show how this method may lead to a better understanding of the biological and pathological significance of this enigmatic property of proteins.