The first analytical method is presented for the identification and absolute configuration determination of all 24 aldohexose and 2-ketohexose isomers, including the D and L enantiomers for allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, and tagatose. Two unique fixed ligand kinetic method combinations were discovered to create significant enough energetic differences to achieve chiral discrimination among all 24 hexoses. Each of these 24 hexoses yields unique ratios of a specific pair of fragment ions that allows for simultaneous determination of identification and absolute configuration. This mass spectrometric-based methodology can be readily employed for accurate identification of any isolated monosaccharide from an unknown biological source. This work provides a key step towards the goal of complete de novo carbohydrate analysis.