The guided gliding of cytoskeletal filaments, driven by biomolecular motors on nano/microstructured chips, enables novel applications in biosensing and biocomputation. However, expensive and time-consuming chip production hampers the developments. It is therefore important to establish protocols to regenerate the chips, preferably without the need to dismantle the assembled microfluidic devices which contain the structured chips. We here describe a novel method toward this end. Specifically, we use the small, nonselective proteolytic enzyme, proteinase K to cleave all surface-adsorbed proteins, including myosin and kinesin motors. Subsequently, we apply a detergent (5% SDS or 0.05% Triton X100) to remove the protein remnants. After this procedure, fresh motor proteins and filaments can be added for new experiments. Both, silanized glass surfaces for actin-myosin motility and pure glass surfaces for microtubule-kinesin motility were repeatedly regenerated using this approach. Moreover, we demonstrate the applicability of the method for the regeneration of nano/microstructured silicon-based chips with selectively functionalized areas for supporting or suppressing gliding motility for both motor systems. The results substantiate the versatility and a promising broad use of the method for regenerating a wide range of protein-based nano/microdevices.