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Determining the sequence and structure of peptides is crucial for understanding their structure–property relationships. Among many techniques, structures are typically elucidated using nuclear magnetic resonance spectroscopy and single crystal X-ray diffraction measurements. In this study, we present terahertz time-domain spectroscopy (THz-TDS) as a complementary, nondestructive technique that is sensitive to both the primary and secondary structures of tetrapeptides. Using only a few milligrams of peptide, THz-TDS spectra have been measured, some of which have been supported by density functional theory (DFT) calculations, to distinguish six tetrameric peptides with similar primary and secondary structures.