Accurate determination of protein structure at interfaces is critical for understanding protein interactions, which is directly relevant to a molecular-level understanding of interfacial proteins in biology and medicine. Vibrational sum frequency generation (VSFG) spectroscopy is often used for probing the protein amide I mode, which reports protein structures at interfaces. Observed peak shifts are attributed to conformational changes and often form the foundation of hypotheses explaining protein working mechanisms. Here, we investigate structurally diverse proteins using conventional and heterodyne-detected VSFG (HD-VSFG) spectroscopy as a function of solution pH. We reveal that blue-shifts of the amide I peak observed in conventional VSFG spectra upon lowering the pH are governed by the drastic change of the nonresonant contribution. Our results highlight that connecting changes in conventional VSFG spectra to conformational changes of interfacial proteins can be arbitrary, and that HD-VSFG measurements are required to draw unambiguous conclusions about structural changes in biomolecules.