In search for enzyme inhibitors, we often encounter "promiscuous" enzyme inhibitors exhibiting nonspecific binding property toward enzyme active site. Therefore, inhibitory candidates should be mechanistically characterized as early as possible in discovery processes. However, there remains a lack of highly reliable and readily available methodology to evaluate specificity of initial hits inhibitors. The present study developed and established a novel DMSO-perturbing assay to identify promiscuous enzyme inhibitors. The assay successfully identified nonspecific binding inhibitors with a broad scope, typically by the attenuation of inhibitory activity by the influence of DMSO-addition. This attenuation would be attributed to the nonspecific binding property of inhibitors toward both productive and nonproductive (nondenatured) states of enzymes in perturbation solution. This working hypothesis was supported by spectroscopic analyses of enzyme conformations and analyses of solvent effects on perturbation. Overall, these results provided a novel concept of the DMSO-perturbing assay.