The interior of the living cell is highly concentrated and structured with molecules having different shapes and sizes. However, almost all experimental biochemical data have been obtained working in dilute solutions that do not reflect in vivo conditions. In this paper, we study in vitro the effect of macromolecular crowding on the reaction rates of the oxidation of 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonate) (ABTS) by hydrogen peroxide (H2O2) catalyzed by Horseradish Peroxidase (HRP), by adding dextrans of various molecular weights to the reaction solutions as crowding agents. The results indicate that the volume occupied by the crowding agent, regardless its size, plays an important role in the rate of this reaction. Both Michaelis-Menten parameters, vmax and Km, decrease when the dextran concentration in the sample increases, which might be due to a crowding-induced effect in the catalytic constant, kcat of this enzymatic reaction. Thus, our results suggest that there is an activation control of the enzymatic reaction in this particular system. In our opinion, this work could facilitate the understanding of biomolecules behavior in vivo and be useful for biotechnology in vitro applications, since HRP is widely used in the development of biosensors.