We present a new assay based on total internal reflection fluorescence (TIRF) to quantify the catalytic activity of adsorbed enzyme monolayers on macroscopically flat surfaces. The need for such an assay derives from a general shortage of assay methods that are sufficiently sensitive to measure reaction kinetics for just a single monolayer of enzymes. The assay is based on the enzymatic conversion of a soluble, nonfluorescent fluorogenic substrate reagent to a soluble, highly fluorescent product. The reaction occurs at the solid-liquid interface where the enzymes are adsorbed. Fluorogenic substrates are introduced to the adsorbed layer by convective diffusion from solutions undergoing steady laminar slit flow. The exponentially decaying evanescent wave that is produced by total internal reflection serves as a "spectroscopic ruler" to resolve the spatial concentration profile of fluorescent products in solution near the interface. By measuring the steady-state fluorescence signal as a function of the Peclet number that characterizes mass transfer conditions in the experiment, it is possible to determine the enzymatic reaction rate. Here we present the development of the method and its application to a test system of beta-galactosidase adsorbed to methylated silica surfaces. Compared to the enzymatic rate constants for this enzyme in free solution, adsorption decreased the Michaelis-Menten rate constant k(cat) by a factor of 10 and increased the equilibrium binding constant K-m, by a factor of 4.5. Thus the intrinsic activity of the enzyme, as represented by the ratio k(cat)/K-m, decreased 45-fold due to adsorption,