In the present study, the adduct formation equilibria of rifampicin (RIF) with cysteine (CySH) and glutathione (GSH) at different pHs were investigated using cyclic and square-wave voltammetry techniques. With increasing RIF concentration, the reduction currents of mercurous thiolates (Hg2(RS)2) which are main surface products for CySH and GSH on Hg electrode decrease drastically and their reduction potentials shifted towards less negative values due to intermolecular interactions. At the same time, the voltammetric signals (especially the Epa and Epc values of reversible redox process of the α-hydrojuglone moiety) of RIF shifted towards positive values in the presence of CySH or GSH. A plausible mechanism is proposed to explain the formations of some adducts by means of this interaction process. That is that CySH and GSH behave as nucleophile and undergo the addition reaction with RIF. The formation of adducts was also confirmed by FTIR measurements and could be clarified by the disappearance of the stretching band of free thiol group. The stoichiometric ratio of this reaction was found to be 1 : 1 and the binding constants were evaluated using the experimental data of square-wave voltammetry. The pH effect on binding constant was studied by varying pH (4.5, 7.4, 9.0). It was found that the reaction was strongly influenced by the pH. This nucleophilic addition reaction was mostly suitable at pH 9. Also, the constant of the addition reaction of GSH is higher than that of CySH for all studied pHs. The thermal behaviours of these adducts were characterized by thermal analysis techniques (TGA/DrTGA/DTA). Since the thermograms recorded for adducts are not an overlapping of thermal curves of starting materials, this behaviour has been considered a further proof for the formation of adducts.