We have measured a vibrationally-resolved UV spectrum of doubly-protonated gramicidin S (GS) in the gas phase and, subsequently, a highly-resolved, conformer-specific infrared spectra in the 6 μm fingerprint region, using a cold ion trap in combination with table-top lasers. The study reveals at least three conformational states of GS populated under our experimental conditions, with the major one showing evidence of a symmetric 3-D structure, similar to that in the condensed phase. The derived qualitative constraints, along with the measured vibrational frequencies, serve as a benchmark for computations of peptide structure.