Since the early days of cell volume regulation research, the role of actin cytoskeleton organization and rearrangement has attracted specific interest. Rapid modifications in actin dynamics and architecture have been described. They were shown to regulate cell volume changes, as well as regulatory volume decrease in a large variety of cell types, including hepatocytes, lymphocytes, fibroblasts, myocytes, and various tumor cells. Using microscopic and biochemical analyses, modifications of actin organization and polymerization dynamics were studied. This chapter summarizes the molecular approaches applied so far for the quantitative assessment of actin cytoskeleton dynamics in the various cell types. It demonstrates that rapid modifications of actin cytoskeleton dynamics regulated by specific signaling pathways play a functional role in cell volume regulation. It is concluded that studying actin polymerization dynamics and signaling represents a challenging tool for the understanding of osmosensing and osmosignaling regulation in cellular physiology.