The great potential of nonlinear optical molecular probing method in accessing molecular level information about adsorption and reaction mechanism at solid–liquid interfaces, under real chemical condition, is demonstrated using the results of our experiments on the adsorption behavior of thionin+ cations at the silica/CH3CN interface. The study has revealed that thionin+ cations are deprotonated due to their strong interactions with the SiO− groups at the interface, thus generating more surface silanol groups (SiOH) and a neutral species thionin0, which stays in the liquid phase due to the lack of strong interaction with the silica substrate.