Mechanobiology, as an evolving field, explores how cells sense and respond to mechanical forces within their microenvironment, particularly the extracellular matrix (ECM). These mechanical cues are essential for regulating several biological processes such as growth, migration, and differentiation, and their dysregulation is linked to diseases including cancer, fibrosis, and developmental disorders. Cell–substrate interaction assumes a central role in mechanobiology. This chapter provides a comprehensive overview of the biophysical methods used to investigate cell–substrate interaction. It delves into key cellular mechanisms, including force generation via focal adhesions, rigidity sensing, and force transmission through cytoskeletal structures, as well as the molecular pathways underpinning these processes that drive tissue organization and cellular behavior. Advanced biophysical techniques, such as traction force microscopy, atomic force microscopy, optical and magnetic tweezers, microforce sensing arrays, and fluorescence resonance energy transfer, are introduced to illustrate how cellular forces are measured to dissect the dynamics at the cell–ECM interface. This chapter also highlights the potential of integrating these techniques with high-resolution imaging to unravel the complex relationships governing cell–substrate interactions.