The “binder/tag” approach, which utilizes the interaction of a 7 amino acid peptide SsrA (tag) and a small protein SspB (binder) to reveal protein conformation, has been employed to probe the conformational changes of individual Src family proteins in living cells. My research extends this approach to identify orthogonal binder/tag pairs for multiplexed biosensor imaging, and applies the binder/tag approach to report the activities of Rho family GTPase guanine exchange factors (GEFs).