The folded form of S-Peptide is found to be essential for the activation of RNase-S complex. Herein the folding-unfolding dynamics of S-Peptide and its protonated form in mild acidic conditions are investigated to assess the most favorable folding pathway in physiological conditions. Our results confirm that the pH assisted S-peptide structures could indeed influence on the binding and collapse of the hydrophobic groups, which in turn can modulate the structural stability of S-Peptide α-helix. We affirm that the protonated S-Peptide has its partially and completely folded states which are readily accessible whereas in the case of non-protonated S-Peptide, there exists an energy barrier in attaining the folds. Further, the RNase-S complex formation could be mostly assisted by mild acidic pH and we also confirm that the folding-unfolding pathways of S-Peptide are independent of the pH. © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim