Chiral HPLC coupled to electronic circular dichroism and laser optical rotation detection (HPLC-ECD-OR) permitted the on-line chiroptical characterization of both enantiomers of racemic flavanone (1) as (ECD)(-)(310)-(OR)(-)(670)-flavanone and (ECD)(+)(310)-(OR)(+)(670)-flavanone for the first and second eluted peaks, respectively. Calculation of the ECD spectrum of one enantiomer at the TD-DFT/DGTZVP level of theory yielded the (S)-(ECD)(-)(310)-(OR)(-)(670) and (R)-(ECD)(+)(310)-(OR)(+)(670)-1 absolute configuration, in agreement with the use of empirical rules for stereochemical assignment. Vibrational circular dichroism spectra of enantiopure compounds, obtained through fraction collection during repeated HPLC runs, were recorded and compared with theoretical traces produced from DFT calculations at the B3LYP/DGDZVP and B3PW91/DGDZVP levels of theory, which further confirmed the absolute configuration obtained from the on-line chiroptical data.