Horseradish peroxidase (HRP), a well-known oxidase, is frequently used in the diagnostic field as a labeling enzyme, where it amplifies the substrate binding signal of antibodies. Though practical for detecting moderate amounts of substrate, there is strong demand for further development of its sensitivity to detect minuscule quantities of biomarkers. Recently, we found that betaine-type cellular metabolite analogs facilitate enzymatic hydrolysis just by dissolving them into the reaction buffer. In the present study, using the analog (2-(N,N,N-tri-n-butylammonium) acetate) and various colorimetric substrates of HRP, we investigated the activation behavior of HRP. As a result, the analog structure- and concentration-dependently facilitated the various HRP-catalyzed oxidative reactions. Interestingly, the analog facilitated not only the reaction rate but also the chromogenic sensitivity. Kinetic and structural analyses revealed that the increased chromogenic sensitivity is related to the enhancement of the conformational flexibility in the substrate binding site in HRP by addition of the analog, which diminishes the binding affinity between HRP and large substrates. The finding serves to create practical applications of the analogs for increased detection sensitivity of HRP-related clinical agents.