Cofactors are organic molecules, most of them originating from vitamins, that bind to enzymes making them able to catalyze defined reactions. A cofactor-based chemogenomics approach exploits the presence of a cofactor-binding domain to develop compound scaffolds tailored to mimic the cofactor and to replace it within target enzyme classes. As a result, a loss of function is observed. An expansion of the cofactor scaffold to include structural/chemical features derived from the substrate, that usually binds at cofactor adjacent sites, increases the specificity of the enzyme fishing. This approach has been so far applied only to NAD(P)(+)-dependent enzymes. However, it is suitable for all other cofactors, with difficulties, for some of them, originated by very tight binding. In the case of cofactors covalently bound to the enzyme, the competition between the natural cofactor and the cofactor scaffold mimic can only occur during enzyme folding.