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Abstract Raman spectroscopy provides label‐free, specific analysis of biomolecular structure and interactions. It could have a greater impact with improved characterization of complex fingerprint vibrations. Many Raman peaks have been assigned to cholesterol, for example, but the molecular vibrations associated with those peaks are not known. In this report, time‐dependent density functional theory calculations of the Raman spectrum of cholesterol are compared to measurements on microcrystalline powder to identify 23 peaks in the Raman spectrum. Among them, a band of six peaks is found to be sensitive to the conformational structure of cholesterol's iso‐octyl chain. Calculations on 10 conformers in this spectral band are fit to experimental spectra to probe the cholesterol chain structure in purified powder and in phospholipid vesicles. In vesicles, the chain is found to bend perpendicular to the steroid rings, supporting the case that the chain is a dynamic structure that contributes to lipid condensation and other effects of cholesterol in biomembranes. Statement of Significance: Here we use density functional theory to identify a band of six peaks in cholesterol's Raman spectrum that is sensitive to the conformational structure of cholesterol's chain. Raman spectra were analyzed to show that in fluid‐phase lipid membranes, about half of the cholesterol chains point perpendicular to the steroid rings. This new method of label‐free structural analysis could make significant contributions to our understanding of cholesterol's critical role in biomembrane structure and function. More broadly, the results show that computational quantum chemistry Raman spectroscopy can make significant new contributions to molecular structure when spectra are interpreted with computational quantum chemistry.