The polarity pattern of a macromolecule is of utmost importance to its structure and function. For example, one of the main driving forces for protein folding is the burial of hydrophobic residues. Yet polarity remains a difficult property to measure experimentally, due in part to its nonuniformity in the protein interior. Herein, we show that Fourier transform infrared (FTIR) linewidth analysis of noninvasive 1-¹³C¹⁸O labels can be used to obtain a reliable measure of the local polarity, even in a highly multiphasic system, such as a membrane protein. We show that in the Influenza M2 H⁺ channel, residues that line the pore are located in an environment that is as polar as fully solvated residues, while residues that face the lipid acyl chains are located in an apolar environment. Taken together, FTIR linewidth analysis is a powerful, yet chemically nonperturbing approach to examine one of the most important properties in proteins: polarity.