The last years, there is a steadily growing demand for methods and materials appropriate to create patterns of biomolecules for bioanalytical applications. Here, a photolithographic method for patterning biomolecules onto a silicon surface coated with a polymeric layer of high protein binding capacity is presented. The patterning process does not affect the polymeric film and the activity of the immobilized onto the surface biomolecules. Therefore, it permits sequential immobilization of different biomolecules on spatially distinct areas on the same solid support. The polymeric layer is based on a commercially available photoresist (AZ5214) that is cured at high temperature in order to provide a stable substrate for creation of protein microarrays by the developed photolithographic process. The photolithographic material consists of a (meth)acrylate copolymer and a sulfonium salt as a photoacid generator, and it is lithographically processed by thermal treatment at temperatures <or=50 degrees C, development with dilute aqueous basic developer solutions and exposure at wavelengths above 300 nm. Following this photolithographic procedure onto the polymeric layer coated silicon surface, protein spots with diameters ranging from 2 to 50 microm were created. The proposed methodology provided good intra-spot homogeneity (CV <or=5%) and inter-spot repeatability (CV <or=5%), as it was determined through epifluorescence microscopy after reaction of the immobilized proteins with their respective fluorescently labeled binding counterparts. Moreover, the polymeric film selected for immobilization of biomolecules presented high protein binding capacity, which was at least three folds higher than that obtained using aminosilanized surfaces. The proposed methodology is expected to facilitate considerably the fabrication of dense protein microarrays for bioanalytical applications.