We report on a facile diffusion-based photopatterning technique for generating linear and non-linear decreasing pore-size gradients in cross-linked polyacrylamide gels. Diffusion of low viscosity polymer precursor solutions and a two-step photopatterning process were used to define the decreasing pore-size gradient gels in a microfluidic format, thus eliminating the need for controlled mixing and delivery of polymer precursor solutions. We present an analytical model of the non-steady state diffusion process and numerically evaluate that model for direct comparison with empirical characterizations of the gradient gels. We show that the analytical model provides an effective means to predict the steepness and linearity of a desired gradient gel prior to fabrication. To assess electrophoretic assay performance in the microfluidic gradient gels, on-chip sizing of protein samples (20–116 kDa) was investigated. Baseline resolution of six proteins was demonstrated in 4 s using 3.5% to 10% polyacrylamide gradient gels. The demonstrated ability to conduct efficient protein sizing in ultra-short separation lengths (0.3 cm) means low applied electric potentials are needed to achieve the electric field strengths required for protein separations. The low required electric potentials relax operating constraints on electrical components, as is especially important for translation of the assay into pre-clinical and clinical settings. The gradient gel fabrication method reported is amenable to adaptation to non-sizing protein assays, as well as integration with upstream sample preparation steps and subsequent orthogonal downstream assays.