This chapter summarizes practical methods for the investigation of nucleotide interactions with GTPases and shows how these can be extended to examine the interaction of such proteins with partner molecules. GTP-binding proteins constitute a class of proteins which are involved in signal transduction and regulation of many processes in the cell. Many GTPases are isolated as stable complexes with GDP, regardless of whether they are purified from their natural source or after overexpression in a heterologous system. Depending on the stability of this complex, this may present a significant problem for both equilibrium and kinetic investigations. Measurements of the kinetics of GDP dissociation from strongly binding GTPases is relatively straightforward and can be accomplished using filter binding or spectroscopic methods. Both intrinsic and extrinsic fluorescence have been used as monitors of the interaction of GTPases with nucleotides. GTPases typically interact with an effector molecule, which is the next molecule in the signal transduction chain.