We constructed a molecular platform to evaluate a tug-of-war between motor proteins, kinesin and dynein. It is the molecular system using the gliding assay, where microtubules (MTs) glide on immobilized motors. In this particular study, we selectively patterned kinesin and dynein on a substrate so as to attach MTs at the boundary between the two motor proteins. We could evaluate the time course of MT position and velocity at the boundary. For the analysis of the number of kinesin and dynein bound to MT, we measured the density of kinesin and dynein on a substrate. We monitored the velocity change of a MT according to the numbers of kinesin and dynein molecules propelling the MT. Force balance between two segments of the MT pulled by kinesin or dynein varies due to the increase or decrease of motors bound. Therefore, we are able to conclude that the velocity change was caused by the force balance between the segments. In conclusion, we could propose a new method to evaluate a tug-of-war between kinesin and dynein.