We combine a surface chemistry approach with a FRET-based biosensor to investigate the effect of changes in the extracellular microenvironment on the spatio-temporal dynamics of RhoA activation in cell protrusions. This approach is based on the use of microcontact printing to pattern self-assembled monolayers of alkanethiolates on gold, to generate cell adhesive and inert regions on the model surface, and the development of a compatible high-resolution fluorescence microscope that overcomes the intrinsic quenching of low concentration and intensity of fluorophores in live cells by the gold surface. Mouse embryonic fibroblasts expressing the RhoA biosensor, confined within the cell adhesive pattern, periodically extend protrusions to sample the inert region of the monolayer outside the pattern. We observed for the first time that RhoA activity is elevated at the leading edge of protrusions in the absence of substrate adhesion.