Biomimetic biomaterials, including hydrogels, are used for in vitro three-dimensional cell cultures with the overall goal to mimic the native extracellular matrix (ECM) of the cells and to investigate cellular mechanotransduction mechanisms induced by the physical microenvironment and stimuli surrounding the cells. Indeed, the mechanical properties of the surrounding environment influence the cellular behavior and functions and can trigger pathological processes. Therefore, characterizing the mechanical properties of biological tissues and proposed biomimetic substrates at a cellular length scale is gaining significant importance in mechanotransduction investigations. The general aim of this work was to support the design of gelatin-methacryloyl (GelMA) hydrogels developed for mimicking in vitro the human healthy and pathological lung tissue, and in particular to characterize by nanoindentation method the mechanical properties of human healthy and pathological lung tissue and GelMA hydrogels.