The characterization of motor proteins is important to the fields of biology, engineering, and medicine as they play a vital role in cellular processes, such as intercellular transport; are linked to many neurological disorders; and serve as an excellent model for nanoscale motors. The present study characterizes kinesin motility with respect to microtubules. Total internal fluorescence microscopy (TIRFM) was used to visualize the motion of single molecules of kinesin along microtubules in varying concentrations of ATP. TIRFM was also used to visualize the motion of microtubules over static kinesin. Data was analyzed using Spot Tracker in tandem with Image J software and custom written image analysis algorithms. Real-time imaging revealed preferred kinesin binding to kinesin decorated microtubules as well as the imposition of significant flexural strain on the microtubules via kinesin. Kinesin velocity was determined to be 0.442±0.079?m/s at 1mM ATP and 0.439±0.112 at 300?M ATP. The knowledge gleaned from this study will assist in the development of diagnosis and treatment procedures for neurological diseases such as Alzheimer’s, expand current models of the cell, and will provide a platform for the fabrication of nanoscale motor devices.