Polyacrylamide gels functionalized with extracellular matrix (ECM) proteins are commonly used as cell culture platforms to evaluate the combined effects of ECM composition, cell geometry and substrate rigidity on cell physiology. For this purpose, protein transfer onto the surface of polyacrylamide hydrogels must result in geometrically well-resolved micropatterns with homogeneous protein distribution. Yet the outcomes of micropatterning methods have not been pairwise evaluated against these criteria. We report a versatile photoresist lift-off patterning (LOP) method to pattern ECM proteins on polyacrylamide hydrogels ranging from 5 to 25 kPa. We directly compare the protein transfer efficiency and pattern geometrical accuracy of this protocol to the widely used microcontact printing (μCP) method. LOP achieves higher protein transfer efficiency, increases pattern accuracy, and reduces variability of these factors within arrays of patterns as it bypasses the drying and transfer steps of microcontact printing. We demonstrate that lift-off patterned hydrogels successfully control cell size and shape when we culture epithelial cells on these platforms. Note: Jens Moeller and Aleksandra K. Denisin contributed equally.