Living cells are constantly exposed to mechanical stimuli arising from the surrounding environment. The ability of a cell to transform stimuli into a biological response, known as mechanotransduction, is crucial in guiding cell behavior. Therefore, the mechanical properties of the extracellular matrix (ECM) determine, synergistically with other stimuli, cell fate, homeostasis, and disease. In this context, gelatin methacryloyl (GelMA) hydrogels have attracted significant attention in the field of tissue engineering and regenerative medicine due to their ability to mimic the native ECM, their excellent biocompatibility, and their tunable physicochemical properties. The main purpose of this study was to mechanically characterize various compositions of GelMA hydrogel, differing in methacrylation degree and GelMA concentration, at the cellular scale.