We utilize single-molecule enzymology to study the distribution of heterogeneous enzyme activity in a sample. To monitor enzymatic activity with single-molecule resolution, we perform the enzyme activity assay inside femtoliter-sized microwells that can be filled with an enzyme-substrate solution. The fluorescent signal generated from a single enzyme molecule becomes highly concentrated within a microwell and is measured over time using a fluorescence microscope. At low enzyme concentrations, the number of enzyme molecules in a well follows a Poisson distribution: most wells contain either one or zero enzyme molecules, and a negligible number of wells contain more than one enzyme molecule. Analyzing an array of hundreds to thousands of microwells simultaneously enables data collection on a sample’s distribution of the enzymatic activity. We are interested in both fundamental and applied aspects of single molecule enzymology: monitoring enzymatic activity at the single-molecule level for diagnostic applications, developing time tolerance therapeutics, and studying how the microenvironment affects the activity of single enzyme molecules.