Physical forces and mechanical properties have critical roles in cellular function, physiology and disease. Over the past decade, atomic force microscopy (AFM) techniques have enabled substantial advances in our understanding of the tight relationship between force, mechanics and function in living cells and contributed to the growth of mechanobiology. The objective of this workshop will be to provide a comprehensive overview of the use of AFM-based force spectroscopy (AFM-FS) to study the elastic properties and dynamics of living cells, expressing exogenous membrane or cytoskeleton modifying proteins. We’ll first introduce the importance of force and mechanics in cell biology and the general principles of AFM-FS methods. We’ll describe procedures for sample and AFM probe preparations, the various AFM-FS modalities currently available and their respective advantages and limitations, the integration of AFM imaging with optical microscopy. We also provide details and recommendations for best usage practices, and discuss data analysis, statistics and reproducibility. We exemplify typical AFM-FS experiments in focusing on live cells over-expressing either the HIV-1 structural P55Gag polyprotein, which assemble and bud at the cell plasma membrane to produce viral particles, or the membrane curving I-BAR protein IRSp53, which promotes membrane protrusions by interacting with actin cytoskeleton and help HIV-1 budding. Fluorescent viral and cellular proteins localized at the cell membrane will be used to correlate AFM force mapping with protein localization with the cell. At the end of this workshop, the participants will be able to understand the basic functioning of a modern bio-AFM and the grand challenges in the area for the next decade