Hydrogels are polymer-based materials with a high water content. Due to their biocompatible and cell-friendly nature, they play a major role in a variety of biotechnological applications. For many of these applications, diffusibility is an essential property influencing the choice of material. We present an approach to estimate diffusion coefficients in hydrogels based on absorbance measurements of a UV area imaging system. A microfluidic chip with a y-junction was employed to generate a fluid-hydrogel interface and the diffusion of lysozyme from the fluid into the hydrogel phase was monitored. Employing automated image and data processing, analyte concentration profiles were generated from the absorbance measurements and fits with an analytical solution of Fick's second law of diffusion were applied to estimate diffusion coefficients. As a case study, the diffusion of lysozyme in hydrogels made from different concentrations (0.5-1.5% (w/w)) of an unmodified and a low-melt agarose was investigated. The estimated diffusion coefficients for lysozyme were between 0.80 ± 0.04×10 m s for 1.5% (w/w) low-melt agarose and 1.14 ± 0.02×10 m s for 0.5% (w/w) unmodified agarose. The method proved sensitive enough to resolve significant differences between the diffusion coefficients in different concentrations and types of agarose. The microfluidic approach offers low consumption of analyte and hydrogel and requires only relatively simple instrumentation.