Thiolate monolayer, protecting a gold nanocluster, is responsible for its chemical behavior and interaction with the environment. Understanding the parameters that influence the stability and reactivity of the monolayer will enable its precise and controlled functionalization. Here we present a protocol for the investigation of the monolayer reactivity in Au25(SR)18 based on MALDI mass spectrometry and NMR spectroscopy. Thiol exchange reaction between cluster and thiol molecules has been investigated showing how this reaction is affected by several factors (stability of the thiols in solution, the affinity of the sulfur to the gold cluster, intermolecular interactions within the ligand layer, etc.). Furthermore, intercluster thiol exchange has been clarified to occur during collisions between particles without thiol release to the solution. In this reaction, the stability of the thiols in solution and the affinity of the sulfur to the gold for the two thiols do not affect the equilibrium position because for both thiols one S–Au bond is broken and one is formed within the cycle. Importantly, the rate of direct thiol exchange between clusters is comparable to that of the ligand exchange with free thiols. However, the thermodynamic driving force of the two reactions is different, since only the latter involves free thiol species.