The mechanical properties of cells have been shown to be tightly correlated to many biological events (such as embryonic development, cell differentiation, cancer metastasis, aging, and disease progression). However, the interplay between the mechanical properties of cells, as well as internal forces and their importance in cell signaling have been insufficiently investigated. A detailed understanding of the effects of cell deformability on cellular and subcellular functions may provide insight into cell signaling and fate. Biophysical techniques such as: (1) atomic force microscopy (AFM); (2) micropipette aspiration; (3) optical tweezing; (4) deformability cytometry; (5) microfluidic channels; and (6) micro-post arrays have been primarily used to measure the stiffness of the cells, while there has been less focus on the role of cell deformability in its functionality. This collection of “Biophysical Methods in Cell Biology” not only brings together conventional and novel experimental techniques (as well as theoretical models adapted from traditional biophysical methods to characterize cell mechanical properties); also, it welcomes methods in the biophysical area which highlight the association of cell mechanical properties in different biological contexts, their roles in cell physiology and disease development, signal transduction, and biologically-relevant responses.