The ability of a living cell to define its shape, withstand environmental stresses, and move is central to that cell’s identity and function. We have played an active role in developing and using single-cell biophysical approaches to investigate the molecular and biophysical basis of cell structure and mechanics. We apply a wide range of technologies for this purpose, including atomic force microscopy, traction force microscopy, and a variety of optical imaging modalities. We are perhaps most widely known for our use of subcellular laser ablation (femtosecond laser nanosurgery) to dissect the mechanical properties and structural contributions of single cytoskeletal elements in living cells (Kumar et al., Biophysical Journal 2006). For example, we have shown that actomyosin stress fibers are viscoelastic cables and that their load-bearing properties depend strongly on cellular location (Tanner et al., Biophysical Journal 2010). We have also combined laser ablation with fluorescence resonance energy transfer (FRET) tension sensors to spatially map tension in single stress fibers to focal adhesions at the cell-ECM interface (Chang and Kumar, Journal of Cell Science 2013; Chang and Kumar, Scientific Reports 2015). Recently, we combined laser ablation with single-cell micropatterning and mathematical modeling to understand how the mechanical properties of stress fibers are governed by the physical networks in which they reside (Kassianidou et al., PNAS 2017). In addition to these high-resolution methods, we have developed complementary strategies to quickly screen mechanical properties within larger populations (Sen and Kumar, Cellular and Molecular Bioengineering 2009; Guillou, Dahl, Lin et al., Biophysical Journal 2016). Finally, we have actively exploited tools for quantitative control of gene expression to elucidate relationships between protein level/activation and phenotype. For example, we have used orthogonal inducer-promoter systems to create “phase diagrams” that relate Rho and Rac GTPase activation to migratory phenotype (MacKay and Kumar, Integrative Biology 2014). We have also used inducible control of cell migration to pattern cells in 3D matrices (MacKay and Kumar, Soft Matter 2013).