AbstractHistidine is an important natural amino acid, involved in many relevant biological processes, which, because of its physical properties, proved difficult to characterize experimentally in its neutral form. In this work, neutral histidine has been generated in the gas phase by laser ablation of solid samples and its NεH tautomeric form unraveled through its rotational spectrum. The quadrupole hyperfine structure, arising from the existing three 14N nuclei, constituted a site‐specifically probe for revealing the tautomeric form as well as the side chain configuration of this proteogenic amino acid.