Protonation of gossypol Schiff bases (S1 and S2), possessing different numbers of basic N-atoms, was studied using potentiometric, spectroscopic, ESI MS and PM5 methods. Titration of S1 and S2 with HClO₄, monitored by the FT-IR and ¹H NMR, indicated that the change from the enamine-enamine into the protonated imine-imine tautomeric form occurs at different Schiff base–H⁺ ratio. The FT-IR and PM5 results show that for S1 the first protonation step occurs at Schiff base moiety whereas for S2 it is realised at N-atom of the morpholine. The formation of N⁺–H⋯O hydrogen bond between morpholine moieties within S2 contributes to high pK_a(ACN) = 22.65.