Atomic force microscopy is an essential tool for cell biology research. It can provide 3D topographical data of living, unfixed cells. However, AFM’s greatest strength in cell biology is its ability to provide accurate and quantitative mechanical measurements in near-physiological conditions (i.e. in culture medium and at 37°C). The elastic and viscoelastic response of a cell or substrate can be routinely measured using force maps and AFM-based microrheology techniques, respectively. The measured cell moduli can be that of unaltered cells, cells in different states of development, differentiation, or disease, or cells responding to a stimulus such as a drug or mechanical stress. Measuring the moduli of substrates and the cell microenvironment is also important due to the role the extracellular matrix (ECM) plays in such processes as cell differentiation, fate, signalling, gene transcription, cancer, cardiovascular disease and apoptosis.