Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes the processive motility assay for investigating the motility of myosin V in vitro. Biotin-Alexa actin filaments are fixed to a slide by biotin/streptavidin linkages and aligned with the microscope x-axis by fluid flow. The orientation of a rhodamine-calmodulin (CaM) probe bound to a single myosin V molecule is determined as it moves along an actin filament. Excess wild-type calmodulin (WT-CaM) is present in the buffer solution to replenish lost CaM from the myosin lever arm. The techniques for myosin V should be generally applicable to other single-molecule experiments where angular changes have an important mechanistic role in their biological function.