Rho GTPases are important regulators of the formation of focal adhesions and focal complexes, and thereby they are key regulators of cell adhesion and migration. Here, we describe a method to study the relocalization of proteins within cell–matrix adhesions at a nanoscale level, through the use of super-resolution stimulated emission depletion microscopy imaging. We furthermore describe computational tools for image processing and data analysis. Thus, the method presented in this chapter provides an unbiased approach to the quantitative evaluation of the spatial distribution of the nanoscale protein assemblies by which cells adhere to an underlying substrate.