This paper reports a method to characterize the kinetic constants for the action of enzymes on immobilized substrates. This example uses cutinase, a serine esterase that hydrolyzes 4-hydroxyphenyl valerate moieties that are immobilized on a self-assembled monolayer of alkanethiolates on gold. The product of the enzyme reaction is a hydroquinone, which is redox active and therefore permits the use of cyclic voltammetry to monitor the extent of reaction in situ. A kinetic model based on the Michaelis-Menten formalism is used to analyze the dependence of initial rates of reaction on both the substrate density and the enzyme concentration. The resulting value of k(cat)/K-M for the interfacial reaction is comparable to that for a homogeneous phase reaction with a substrate of similar structure. This strategy of using monolayers presenting substrates for the enzyme and cyclic voltammetry to measure reaction rates provides quantitative and real-time information on reaction rates and permits a level of analysis of interfacial enzyme reactions that to date has been difficult to realize.