Nanoparticle surface coatings dictate their fate, transport, and bioavailability. We used a gold nanoparticle-bacterial cellulose substrate and "hot spot"-normalized surface-enhanced Raman scattering (HSNSERS) to achieve in situ and real-time monitoring of ligand exchange reactions on the gold surface. This approach enables semiquantitative determination of citrate surface coverage. Following exposure of the citrate-coated nanoparticles to a suite of guest ligands (thiolates, amines, carboxylates, inorganic ions, and proteins), the guest ligand signal exhibited first-order growth kinetics, while the desorption mediated decay of the citrate signal followed a first-order model. Guest ligand functional group chemistry dictated the kinetics of citrate desorption, while the guest ligand concentration played only a minor role. Thiolates and BSA were more efficient at ligand exchange than amine-containing chemicals, carboxylate-containing chemicals, and inorganic salts due to their higher binding energies with the AuNP surface. Amine-containing molecules overcoated rather than displaced the citrate layer via electrostatic interaction. Citrate exhibited low resistance to replacement at high surface coverages, but higher resistance at lower coverage, thus suggesting a transformation of the citrate-binding mode during desorption. High resistance to replacement in streamwater suggests that the role of surface-adsorbed citrate in nanomaterial fate and transport must be better understood.