The lateral mobility of the thiolate ligands on the surface of Au nanoparticles was probed by EPR spectroscopy. This was achieved by using bisnitroxide ligands, which contained a disulfide group (to ensure attachment to the Au surface) and a cleavable ester bridge connecting the two spin-labeled branches of the molecule. Upon adsorption of these ligands on the surface of Au nanoparticles, the two spin-labeled branches were held next to each other by the ester bridge as evidenced by the spin-spin interactions. Cleavage of the bridge removed the link that kept the branches together. CW and pulsed EPR (DEER) experiments showed that the average distance between the adjacent thiolate branches on the Au nanoparticle surface only marginally increased after cleaving the bridge and thermal treatment. This implies that the lateral diffusion of thiolate ligands on the nanoparticle surface is very slow at room temperature and takes hours even at elevated temperatures (90 degrees C). The changes in the distance distribution observed at high temperature are likely due to ligands hopping between the nanoparticles rather than diffusing on the particle surface.