Proteins are built of amino acid residues that differ from each other only by their side chain. The pH-dependent response of these side chains on protein surface is vital for various biological processes. Here we have investigated the aqueous interface in the presence of different amino acid side chains at varying pH (bulk) using heterodyne-detected vibrational sum-frequency generation (HD-VSFG) spectroscopy. It is observed that amine/imidazolic (e.g., lysine/histidine) and alcoholic (e.g., serine and threonine) side chains preferentially orient the interfacial water as “H-down” (i.e., the water hydrogens are pointed toward the aqueous bulk) in acidic solution (pH ∼2). At physiological pH (7.4), the interfacial water takes “H-up” orientation (i.e., the water hydrogens are pointed away from the aqueous bulk) for the alcoholic/imidazolic side chains but remains “H-down” for the amine containing side chain. On further increasing the pH (up to 12.0), the interfacial water becomes increasingly H-up oriented, revealing the charging of the interface for all side chains investigated. Because the side chains are uncharged at high pH (12.0), the charging of the aqueous interface implies the adsorption of OH− anion at the interface. Moreover, in the case of imidazolic side chain (i.e., histidine), the flip-flop of interfacial water within a narrow range of pH 6.0−7.5 shows the reversal of interfacial electric field within a little variation of [H⁺] (or [OH⁻]) near the physiological pH. This observation provides direct experimental evidence in support of the hypothesis that the pH-dependent oxygen affinity of hemoglobin (Bohr effect) is due to changing electrostatics of histidine at the surface of hemoglobin.