Rho GTPases, including RhoA, Rac1 and Cdc42, are key regulators of cell migration in animals. In a recent issue of Nature, two papers present delicate studies on precise roles and spatiotemporal coordination of Rho GTPases in live cells, using either a photoactivatable Rac1 or specific biosensors, together with computational multiplexing method. In addition to implications in cell migration, analytical tools and methods used in these works can also be extended into other live cell studies on complex cellular/molecular events.