Rotational spectroscopy in combination with molecular beams and laser ablation (laser-ablation molecular-beam Fourier transform microwave (LA-MB-FTMW) spectroscopy) has proved to be successful in characterizing the conformers of natural amino acids. The procedure usually followed to assign and identify the different conformers of an amino acid from the rotational spectrum is described through the study of the natural amino acidL-threonine. The solid sample of L-threonine was vaporized by laser pulses, diluted in Ne and supersonically expanded between the mirrors of a Fabry–Pérot resonator where it was spectroscopically probed by microwave radiation. The rotational and nuclear quadrupole coupling constants extracted from the analysis of the rotational spectrum are directly compared with those predicted by ab initio methods to achieve the conclusive identification of seven different conformers. A complex hydrogen bonding network arises as a consequence of the polar side chain of threonine.