Here we present our standard protocol for studying the binding of kinetochore proteins to microtubules as a paradigm for designing single-molecule total internal reflection fluorescence (TIRF) microscopy experiments. Several aspects of this protocol require empirical optimization, including the method for anchoring the polymer or substrate to the coverslip, the type and amount of blocking protein to prevent nonspecific protein adsorption to the glass, the appropriate protein concentration, the laser power, and the duration of imaging. Our method uses bovine serum albumin and κ-casein as blocking agents to coat any imperfections in the coverslip silanization and thereby prevent protein adsorption to the coverslip. Protein concentration and duration of imaging must be optimized for each experiment and protein of interest. Ideally, a range is determined that allows for resolution of single complexes binding to microtubules to ensure proper measurement of kinetic off rates and diffusion along microtubules. Excessively high concentrations may lead to overlapping binding of proteins on microtubules, making it impossible to resolve single binding events. The duration of imaging must be long enough to capture very low off rates (long residence time on microtubules) and we typically image at 10 frames/sec for 200 sec. The laser power can be adjusted to prevent photobleaching, but must be high enough to achieve a sufficient signal/noise ratio.