Many fundamental biological processes, including early embryo development, immune responses and the progression of pathogens, are mediated by gradients of biological molecules. Understanding these vital physiological processes requires the development of biomaterial platforms that can mimic them in-vitro. Such platforms include laboratory generated surface gradients of biological molecules. In this work, we report a method for the generation of surface gradients of two proteins. We used a surface grafting density gradient of polyethylene glycol (PEG) to control protein adsorption. In addition, we used protein size as a tool to control the position and the adsorbed amount of both proteins. To demonstrate our concept, we used fibrinogen as an example of a large protein and lysozyme as an example of a small protein. However, we speculate that the same strategy could be extended to any other pair of large and small proteins. We used X-ray photoelectron spectroscopy and sessile drop contact angle measurements to determine the chemical composition and wettability of the gradients. Protein adsorption was studied by surface plasmon resonance imaging.