Genome-wide association studies and genetic linkage studies have created a growing list of proteins related to disease. Small molecules can serve as useful probes of function for these proteins in a cellular setting or may serve as leads for therapeutic development. High-throughput and general binding assays may provide a path for discovering small molecules that target proteins for which little is known about structure or function or for which conventional functional assays have failed. One such binding assay involves small-molecule microarrays (SMMs) containing compounds that have been arrayed and immobilized onto a solid support. The SMMs can be incubated with a protein target of interest and protein-small molecule interactions may be detected using a variety of fluorescent readouts. Several suitable methods for manufacturing SMMs exist and different immobilization methods may be more or less preferable for any given application. Here, we describe protocols for covalent capture of small molecules using an isocyanate-coated glass surface and detection of binding using purified protein.