This protocol describes the techniques to synthesize and fabricate micropatterned poly(ethylene glycol) diacrylate-based hydrogels that can be used as substrates in cellular studies and tissue engineering scaffolds. These materials provide an essentially bioinert background material due to the very low protein adsorption characteristics of poly(ethylene glycol), but the materials can be modified with covalently grafted peptides, proteins, or other biomolecules of interest to impart specific biofunctionality to the material. Further, it is possible to use micropatterning technologies to control the localization of such covalent grafting of biomolecules to the hydrogel materials, thus spatially controlling the cell-material interactions. This protocol presents a relatively simple approach for mask-based photolithographic patterning, generally best suited for patterning the surface of hydrogel materials for 2D cell studies. A more sophisticated technique, two-photon laser scanning lithography, is also presented. This technique allows free-form, 3D micropatterning in hydrogels.