Microbial pathogens may hijack the actin cytoskeleton machinery to facilitate actin-based motility, cellular invasion, and intracellular trafficking through the endocytic pathway. Of particular interest has been a large class of virulence factors collectively termed the type III "effector" proteins. Following contact with a eukaryotic cell, Gram-negative bacterial pathogens employ a dedicated protein secretion apparatus termed type III to translocate effector proteins into the cellular cytoplasm where they initiate a pathogenic response. Interestingly, these effectors can mimic the structure or function of eukaryotic signaling proteins and often target components of the actin cytoskeleton. This chapter describes methodologies to examine the cellular role of the recently defined WxxxE family of effector proteins that functionally mimic Rho family small G proteins.