DiscussionKinesin motor proteins are microtubule-associated proteins that play a key role in fundamental functions such as intracellular transport, motility, cell division and regulation of physiological processes. Therefore, the kinesin family proteins are highly interesting drug targets in the treatment of motor protein related diseases, for example cancer, Alzheimer’s, Huntington’s. Unfortunately, to date there are only few effectors known to act on kinesin motor proteins. This may be due to the fact that, the currently available screening methods are ineffective in detecting the effects of drug candidates on motor proteins. The state-of-the-art is to measure the enzymatic ATPase activity of the motor protein rather than the actual molecular transport function, which the motor performs in the cell. Here we present a high-throughput screening method based on a microtubule-gliding assay, where motors immobilized on a substrate propel microtubules along the surface. We demonstrate that we can measure the speed of such gliding microtubules at high-throughput using a fluorescence microscope and an automated microtubule tracking software. In order to validate the methods efficiency and reliability, we tested different concentrations of a known kinesin-1 inhibitor (AMPPNP) and observed a direct correlation between inhibitor concentration and microtubule gliding velocity.