Rho GTPases, including Rac1, Cdc42, play a critical role in the regulation of a variety of cellular processes such as cell morphology, cell migration, transcriptional activation and gene expression. We constructed several FRET-based single-molecule probes containing red fluorescent protein dsRed1, cyan fluorescent protein ECFP, the GTPase binding domain of the effector, Pak1 or N-WASP, and Rac1 or Cdc42. Rac1 and Cdc42 signaling pathways were activated in transfected cells by the inducer, insulin or bradykinin respectively. In vitro fluorescent spectroscopy assays showed that FRET phenomena were observed in transfected NIH3T3 and Hela cells. For all 3 signaling pathways in NIH3T3 cells, the values of FRET efficiency reached the highest after induction for 5 min, but the increasing extents of the values of FRET efficiency varied in 3 signaling pathways. The values of FRET efficiency decreased with the extention of the induction time, but differed significantly in the decreasing speed for the signaling pathways. Rac1 and Cdc42 activation assays indicated that Rac1 and Cdc42 were in the activated state (GTP-bound) in the induced cells. Their relative activated activities in the cells induced for different time were consistent with the values of FRET efficiency. The activated Rac1, Cdc42 signaling pathways led to the formation of lamelliopodia and filopodia in the transfected cells respectively. The results showed that these single-molecule probes could be used to directly monitor the spatial and temporal imaging of the induced activation of the signaling pathways in living cells. With these single-molecule probes, the GEF or GAP activities of putative regulatory proteins for Rac1 and Cdc42 were analyzed and judged, thus greatly simplifying the currently-used methods for identifying the regulatory proteins for Rho GTPases.