The conformational properties of 2′-fluoro-substituted acetophenone derivatives were elucidated based on H α –F and C α –F through-space spin–spin couplings (TS-couplings), which occur between two atoms constrained at a distance smaller than the sum of their van der Waals radii. This study revealed that 2′-fluoro-substituted acetophenone derivatives in solutions form exclusively s - trans conformers by analyzing their NMR spectra focused on the TS-couplings. The magnitudes of the coupling constants 5 J (H α , F) and 4 J (C α , F) correlate linearly with the value of the dielectric constant of the solvents. Furthermore, s - trans conformations of the two derivatives were confirmed by X-ray crystallographic analysis. These conformational preferences were consistent with the DFT calculations. The s - cis conformer, in which fluorine and oxygen atoms lie in a syn -periplanar mode, may be subject to strong repulsion between the two polar atoms and become unstable. The s - trans preference of the 2′-fluoro-substituted acetophenone derivatives may be utilized in drug design.