Hydrogels are useful platforms as three-dimensional (3D) scaffolds for cell culture, drug-release systems, and regenerative medicine applications. Here, we propose a novel chemical cross-linking approach by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione or diethyl squarate for the preparation of 5 and 10% w/v gelatin-based hydrogels. Hydrogels showed good swelling properties, and the 5% gelatin-based hydrogel proved suitable as a 3D cell culture scaffold for the chondrocyte cell line C28/I2. In addition, diffusion properties of different sized molecules inside the hydrogel were determined.