Single-molecule enzymology allows scientists to examine the distributions of kinetic rates among members of a population. We describe a simple method for the analysis of single-molecule enzymatic kinetics and provide comparisons to ensemble-averaged kinetics. To isolate our model enzyme, alpha-chymotrypsin, into single molecules, we use an array of cylindrical poly(dimethylsiloxane) wells 2 mu m in diameter and 1.35 mu m in height. Inside the wells, a protease assay with a profluorescent substrate detects alpha-chymotrypsin activity. We hold the concentration of alpha-chymotrypsin at 0.39 nM in a given well with an enzyme-to-substrate ratio of 1:6,666 molecules. Fluorescence emitted by the substrate is proportional to enzyme activity and detectable by a charge-coupled device. This method allows for the simultaneous real-time characterization of hundreds of individual enzymes. We analyze single-molecule kinetics by recording and observing their intensity trajectories over time. By testing our method with our current instruments, we confirm that our methodology is useful for the analysis of single enzymes for extracting static inhomogeneity. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 929-937, 2009