Although initially described as major regulators of cytoskeletal remodeling, Rho GTPases have been implicated in the establishment of polarity, endocytosis, vesicle trafficking, morphogenesis, cytokinesis, transcriptional activation, cell cycle progression, and apoptosis, to mention a few. In addition, Rho GTPases have acquired medical relevance because of their participation in tumorigenesis and metastasis, in cardiovascular conditions, and as targets of infectious agents. The field has broadened even more with the contribution of studies in model organisms (plants, amoebas, fungi, invertebrates), each adding a particular view to the complexity of the family and vastly enriching our perception of these important signaling components. Divided into five convenient sections, provides an historical overview of the field and an account of the phylogenetics of the Rho family, general biochemical methods, and functional assays that allow monitoring the consequences of manipulating Rho GTPases in a variety of contexts. Additionally, the volume devotes a section to advanced imaging methods and to recently developed high throughput methods, closing with techniques specifically designed for studies in selected non-mammalian model organisms. Written in the successful series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, provides techniques that are standard for researchers in the field but also includes protocols for those who, being already familiar with some of the techniques, wish to explore additional aspects.