Thioamides are used as potential surrogates of amides to study the structure and dynamics of proteins and nucleic acids. However, incorporation of thioamides in biomolecules leads to changes in their structures and conformations mostly attributed to the strength of the amide-N-H···S═C hydrogen bond. In most cases, it is considered weak owing to the small electronegativity of sulfur, and in some cases, it is as strong as conventional H-bonds. Herein, adopting PDB structure analysis, NMR spectroscopy, and quantum chemistry calculations, we have shown that thioamides in a geometrical and structural constraint-free environment are capable of forming strong H-bonds like their amide counterparts. These studies also enabled us to determine the amide-N-H···S═C H-bond enthalpy (ΔH) very precisely. The estimated ΔH for the amide-N-H···S═C H-bond is ∼-30 kJ/mol, which suggests that the amide-N-H···S═C H-bond is a strong H-bond and merits its inclusion in computational force fields for biomolecular structure simulations to explore the role of amide-N-H···S═C H-bonds in nucleobase pairing and protein folding.