We describe the development of vibrational spectroscopy as a probe of electrostatic driving forces in the formation of stable and functional protein-protein interfaces. Biological function arises from complex interactions between cellular macromolecules, such as the organization of two or more proteins into a functioning assembly. In the post-genomic era, enhanced understanding of the cooperativity between biological molecules is necessary to explore the complexity of living cells. The affinity and specificity of macromolecular interactions are the result of both structural and electrostatic driving forces, but while the field of structural biology has made great advances in this area, much less is understood about electrostatic influences. We are approaching this problem using vibrational Stark effect (VSE) spectroscopy, in which spectral transitions of molecular probes that are placed strategically at a protein-protein interface can be related directly to the strength and direction of electrostatic fields in the immediate vicinity of the probe. Here, we present a vibrational spectroscopic investigation of the electrostatic driving forces that regulate the formation of a stable interface between the oncoprotein Ras and a downstream effector RalGDS. The experimental electrostatic map of this interface obtained through VSE spectroscopy is connected to kinetic and thermodynamic studies of Ras-effector binding, and to preliminary investigations of the interface formed by mutants of Ras that are known to lead to oncogenic function.