The recent development of the DNA-binding domain (DBD)-dynein chimera motors with a dynein motor core and a DNA-binding domain has made it possible to move on DNA nanostructure tracks. In contrast to naturally occurring cytoskeletal filaments such as microtubules and actin filaments, DNA tracks can be programmed with structural properties such as length, stiffness, and circumference. There might be many advantages to using DNA as a track, for example, for applications in nanotechnology. However, care must be taken in design and motility assay conditions to ensure the successful operation of such novel motors; the novel motor system functions under conditions different from those commonly used in the field of natural biomolecular motors. Here, we describe the methods for designing DNA nanostructures and the conditions for motility assays in which the DBD-dynein motors translocate DNA nanotube tracks or move on them.