The functioning of many enzymes is metal ion dependent. In this work, deprotonation/sodiation of gramicidin S (GS) was studied by electrospray ionization mass spectrometry. Electrospray mass spectra for 10−5 M of GS in water/methanol with and without the addition of 1 mM NaOH, AcONa, NaCl, NH4Cl, AcONH4 and 1% AcOH were measured. It was found that one and two amide N–H bonds for [GS + H]+ and [GS + 2H]2+, respectively, were preferably deprotonated to form [GS + H − H + Na]+ and [GS + 2H − 2H + 2Na]2+. The extent of sodiation was found to be in the order of NaOH > AcONa > NaCl. With the addition of 1 mM NaOH, [GS + 2H − nH + nNa]2+ with n up to 8 were observed indicating that all of the eight amide N–H bonds in GS were deprotonated. Sodiation sites are likely to be the negatively charged amide enolate C–O− to form salt-like C–O−⋅⋅⋅⋅Na+ bonds. In the negative mode, one and two amide N–H bonds are preferably deprotonated to form [GS − H]−, [GS − 2H]2− and [GS − 2H + Na]− for the solution with the addition of 1 mM NaOH.