Patterned regular sieves and filters with comparable molecular dimensions hold great promise as an alternative to conventional polymeric gels and fibrous membranes to improve biomolecule separation. Recent developments of microfabricated nanofluidic sieves and filters have demonstrated superior performance for both analytical and preparative separation of various physiologically relevant macromolecules, including proteins. The insights gained from designing these artificial molecular sieves and filters, along with the promising results gathered from their first applications, serve to illustrate the impact that they can have on improving future separation of complex biological samples. Further development of artificial sieves and filters with more elaborate geometrical constraints and tailored surface functionality is believed to provide more promising ideals and results for biomolecule separation, which has great implications for proteomic research and biomarker discovery.