Drug discovery and chemical genetic efforts typically focus on the identification and design of inhibitors or loss-of-function probes as a means to perturb enzyme function. These tools are effective in determining the physiological consequence of ablating the activity of a specific enzyme. Remarkably, nearly a dozen examples of non-natural small molecules that activate enzyme catalysis have been identified within the past decade. In aggregate, these studies delineate four unique activation mechanisms that the small molecules exploit. These complementary gain-of-function probes offer a way to address the sufficiency of an enzyme to drive a particular cellular phenotype, and they also provide new opportunities for drug discovery. This review covers the identification and characterization of these unique small-molecule activators.