New challenges of life science research make nanostructured polymeric surfaces, chemically and spatially resolved, very attractive for biomedical purposes. High costs and unsuitability of polymers processed by the traditional techniques lead to new research routes. In this work, the production of nanostructured polystyrene substrates combining Colloidal lithography and cold plasma processes is shown. Colloidal nanoparticles self-assemble into a regular lattice on the polymers, and are used as physical masks in lithographic processes. Ammonia plasma treatments have been used to improve the suitability of the polymer surface for the entire process. Plasma etching with Ar/O(2) discharges have been applied to transfer the patterns.