We describe a phage display approach to select active Rho-specific scFv sensors. This in vitro technique allows preserving the antigen conformation stability all along the selection process. We used the GTP locked RhoBQ63L mutant as antigen against the Griffin.1 library composed of a human synthetic VH + VL scFv cloned in the pHEN2 phagemid vector. The method described here has permitted to identify an scFv that discriminates between the activated and the inactivated form of the Rho subfamily.