The authors describe a high-throughput patterning process used to create arrays of molecular-scale features for the study of cytoskeletal protein binding interactions. The process uses a shadow-evaporated metal mask to facilitate lift-off of features defined by nanoimprint lithography. This simple and robust approach alleviates difficulties in pattern transfer of ultrasmall features and results in arrays of highly ordered sub-10 nm features which are then functionalized with extracellular matrix proteins. Application of these arrays is demonstrated in cell spreading assays. (C) 2009 American Vacuum Society. [DOI: 10.1116/1.3043472]