A new method is presented for measuring the rate of the oxidation of glucose in the presence of glucose oxidase. The improved method employs luminescence measurements to directly determine the concentration of oxygen in real time, thus obviating complicated reaction schemes employed in previous methods. Our method has been used to determine kinetic parameters including V[subscript max], K[subscript m], and k[subscript cat], all in good agreement with published values. The method can also be used to investigate specificity and inhibition. Information is included pertaining to the construction of a low-cost fluorescence instrument suitable for the measurements in this protocol. (Contains 3 figures.)