Cell response to matrix mechanics is well-known; however, the ability to spatially pattern matrix stiffness to a high degree of control has been difficult to attain. This study describes the use of maskless photolithography as a flexible process for direct, noncontact gradient patterning of photodegradable hydrogels with custom graphics. Any input gray scale image can be used to directly chart hydrogel cross-link density as a function of spatial position. Hydrogels can be patterned with submicron resolution, with length scales within a single substrate spanning several orders of magnitude. A quantitative relationship between input grayscale image pixel intensity and output gel stiffness is validated, allowing for direct gradient patterning. Such physical gradient hydrogel constructs are rapidly produced in a highly controlled fashion with measured stiffness ranges and length scales that are physiologically relevant. Mesenchymal stem cells cultured on these physical gradients matrices congregate and align orthogonal to the gradient direction along iso-degraded lines. This approach results in a robust and high-throughput platform to answer key questions about cell response in heterogeneous physical environments.