Identifying the pKa values of aspartic acid (Asp) and glutamic acid (Glu) in active sites is essential for understanding enzyme reaction mechanisms. In this study, we investigated the correlation between the C═O stretching vibrational frequency (νC═O) of protonated carboxylic acids and the pKa values using density functional theory calculations. In unsaturated carboxylic acids (e.g., benzoic acid analogues), νC═O decreases as the pKa increases (the negative correlation), whereas in saturated carboxylic acids (e.g., acetic acid analogues, Asp, and Glu), νC═O increases as the pKa increases (the positive correlation) as long as the structure of the H-bond network around the acid is identical. The negative/positive correlation between νC═O and pKa can be rationalized by the presence or absence of the C═C double bond. The pKa shift was estimated from the νC═O shift of Asp and Glu in proteins on the basis of the negative correlation derived from benzoic acids. The previous estimations should be revisited by using the positive correlation derived in this study, as demonstrated by quantum mechanical/molecular mechanical calculations of νC═O and electrostatic calculations of pKa on a key Asp85 in the proton-transfer pathway of bacteriorhodopsin.