We report on a novel approach for the size-dependent fractionation of protein assemblies on polymeric surfaces. Using a simple temperature gradient method to generate one-dimensional gradients of grafted poly(ethylene glycol), we fabricated silicon-oxide chips with a gradually changing surface density of kinesin motor molecules. We demonstrate that such a bioactive surface can be used to sort gliding microtubules according to their length. To our knowledge, this is the first example of the self-organized sorting of protein assemblies on surfaces.