This study optimized a micro-contact printing protocol to pattern fibronectin for potential nanoparticle uptake studies using endothelial cells. Different weights were tested during the micro-contact printing process and 20 grams was found to produce the most uniform fibronectin intensity across patterned areas and provided the most accurate coverage of the targeted 2500 μm2 area. This optimized 20 gram weight protocol will be adopted for future studies analyzing how cell cytoskeletal alignments influenced by patterned areas affect nanoparticle uptake in endothelial cells.