<p>Vitamin C (L-( +)-ascorbic acid, L-AA) is an essential micronutrient. Its diastereoisomer, D-(-)-isoascorbic acid (D-IAA), is only 5% as active as L-AA. Therefore, it is crucial to identify and characterize the diastereoisomers of ascorbic acid. In this work, a straightforward and direct method using ion mobility-mass spectrometry (IM-MS) was proposed to identify ascorbic acid isomers. Ternary complexes [L-AA + γ-CD + 2Cs-H]⁺ and [D-IAA + γ-CD + 2Cs-H]⁺, formed by non-covalent interaction of the isomer with the selective agent γ-CD and the metal ion Cs⁺, were separated in ion drift tubes with a resolution of R_<i>p-p</i> as high as 1.398. Meanwhile, comparisons with different CDs and metal ions revealed varying separation efficiencies. Theoretical calculations were conducted to determine the optimal conformations of [L-AA + γ-CD + 2Cs-H]⁺ and [D-IAA + γ-CD + 2Cs-H]⁺. Conformational analysis highlighted distinct structural differences at the molecular level, providing insight into the mobility separation mechanism of AA via the formation of ternary complexes with γ-CD and metal ions. Additionally, a quantitative analysis for the determination of chiral isomers was established with effective linearity and acceptable sensitivity. The method was successfully applied to assess L-AA/D-IAA content in pharmaceuticals and fruit samples.</p> Graphical Abstract <p></p>