Abstract
Rho family GTPases are central regulators of cytoskeletal dynamics controlled by guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). This protocol presents a workflow for a robust high-throughput compatible biosensor assay to analyze changes in Rho GTPase activity by these proteins in the native cellular environment. The procedure can be used for semi-quantitative comparison of GEF/GAP function and extended for analysis of additional modulators. The experimental design is applicable also to other monomolecular ratiometric FRET sensors. For complete details on the use and execution of this protocol, please refer to Müller et al. (2020).