Adsorption of the tripeptide L-glutathione (gamma-glu-cys-gly) on gold surfaces was investigated by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and attenuated total reflection (ATR) infrared spectroscopy. PM-IRRAS was used to study ex situ the adsorbate layer prepared from aqueous solutions at different pH, whereas ATR-IR was applied to study in situ adsorption from ethanol in the presence and absence of acid and base. ATR-IR was furthermore combined with modulation spectroscopy in order to investigate the reversible changes within the adsorbate layer induced by acid and base stimuli, respectively. The molecule is firmly anchored on the gold surface via the thiol group of the cys part. However, the ATR-IR spectra in ethanol indicate a further interaction with the gold surface via the carboxylic acid group of the gly part of the molecule, which deprotonates upon adsorption. Hydrochloric acid readily protonates the two acid groups of the adsorbed molecule. During subsequent ethanol flow the acid groups deprotonate again, a process which proceeds in two distinct steps: a fast step associated with the deprotonation of the acid in the glu part of the molecule and a considerably slower step associated with deprotonation of the acid in the gly moiety. The latter process is assisted by the interaction of the corresponding acid group with the surface. The spectra furthermore indicate a rearrangement of the hydrogen bonding network within the adsorbate layer upon deprotonation. Depending on the protonation state during adsorption of L-glutathione, the response toward identical protonation-deprotonation stimuli is significantly different. This is explained by the ionic state-dependent shape of the molecule, as supported by density functional theory calculations. The different shapes of the individual molecules during layer formation thus influence the structure of the adsorbate layer.