Rho GTPases act as tightly regulated molecular switches governing a large variety of critical cellular functions. Their activity is controlled by two different biochemical reactions, the GDP/GTP exchange and the GTP hydrolysis. These very slow reactions require catalysis in cells by two kinds of regulatory proteins. While the guanine nucleotide exchange factors (GEFs) activate small GTPases by stimulating the exchange of bound GDP for the cellular abundant GTP, GTPase-activating proteins (GAPs) accelerate the intrinsic rate of GTP hydrolysis by several orders of magnitude, leading to their inactivation. There are a number of methods that can be used to characterize the specificity and activity of such regulators to understand the effect of binding on the protein structure and, ultimately, to gain insights into their biological functions. This chapter describes (1) detailed protocols for the expression and purification of Rho GTPases, of -effector-binding domains, and catalytic domains of GEFs and GAPs; (2) the preparation of nucleotide-free and fluorescent nucleotide-bound Rho GTPases; and (3) methods for monitoring the intrinsic and GEF-catalyzed nucleotide exchange, the intrinsic and GAP-stimulated GTP hydrolysis, and the effector interaction with active GTPase (three alternative approaches).