In this work, we present the viability of SERS spectroscopy coupled with isotopically-labeled ligands as a tool for studying monolayers adsorbed onto plasmonically-active surfaces. In particular, the entropy-driven self-exchange of cysteine isotopologues on AgNPs has been studied using SERS spectroscopy. It was concluded that at 25 °C a delay as small as one minute between the introduction of equimolar aliquots of particular isotopologues is sufficient for the formation of a monolayer composed primarily of the first isotopologue added. However, increasing the temperature during the adsorption process changes the composition of the surface monolayer, as it approaches the equilibrium state (in which the ratio of the surface concentrations of isotopologues is equal to the ratio of the amounts of isotopologues within the whole system) at 60 °C. Temperature ramp experiments revealed that the observed monolayer behavior is reversible. The SERS spectrum is unaffected by any temperature changes prior to addition of the second the shape of the marker-bands region is determined by the temperature at which the second isotopologue was added even if the temperature was higher beforehand. The proposed explanation for such an abrupt but reversible change upon temperature increase is a phase transition that occurs in the monolayer. At an elevated temperature, the number of monolayer defects increases, allowing for a dynamic exchange leading to an equilibrium state that was kinetically restricted at 25 °C.