Increasingly accurate mathematical models have been developed to relate solute and hydrogel properties to solute diffusion coefficients in hydrogels, primarily by comparing solute sizes and hydrogel mesh sizes. Here, we use a standardized, high-throughput method for fluorescence recovery after photobleaching (FRAP) experiments and analysis to characterize the diffusion coefficients of fluorescein, three sizes of FITC-dextran, and three sizes of FITC-conjugated poly(ethylene glycol) (PEG) through 18 structurally varied poly(vinyl alcohol) (PVA) hydrogel formulations. Increasing the hydrogel mesh radii increased the diffusivities of all the tested solutes within the hydrogels. While the diffusivity of FITC-dextrans in hydrogels decreased with increasing solute size, the diffusivity of FITC-PEGs increased with increasing solute size, suggesting that a generalized hydrodynamic radius-based model is not universally applicable for solute diffusion in hydrogels. The high-throughput characterization method for solute diffusion in hydrogels described here facilitates precise hydrogel design for biomedical applications.