Gradients of proteins play a prominent role in many biological processes, from development of multicellular organisms to chemical signaling in the immune system. Deposition of surface gradients is a way to permanently modifying a surface’s properties, resulting in the creation of novel materials which have widespread applications in biologically relevant fields, such as directed cell growth, production of biocompatible implantable materials, and creation of functional biosensors. These types of surfaces can also be used as an ex vivo tool to help understand many biological processes by mimicking the environment in gradient-related phenomena in a controllable way. However, despite the large number of applications for chemically graded surfaces, creating them remains a challenge. In this work, a novel diffusion-based patterning mechanism is presented that relies on a 3D micropatterned poly(ethylene glycol) diacrylate (PEG-DA) ‘stamps’ for the controlled deposition of fluorescently-tagged protein ‘ink’ onto pre-treated glass slides. By controlling the contact time and mechanical deformation of the PEG hydrogel on the glass surfaces, it is possible to control local concentration of protein deposition.