Total internal reflection fluorescence microscopy (TIRFM) allows fluorescent molecules on or near the plasma membrane to be visualized with a very high signal-to-noise ratio. This strategy has been very successful for imaging molecular machines as they move and do work. We provide here a general protocol for imaging single molecular motors as they move along microtubule tracks. Our protocol is designed for the study of cytoplasmic dynein purified from Saccharomyces cerevisiae, but it represents a general framework for any in vitro single-molecule assay.