The isomer ratio determination of a selenium-containing metabolite produced by Se-rich yeast was performed. Electrospray ionization and ion mobility mass spectrometry (IM-MS) were unsuccessfully used in order to resolve the isomers according to their collisional cross section (CCS) difference. The isomer ratio determination of 2,3-dihydroxypropionylselenocystathionine was performed after multidimensional liquid chromatography preconcentration from a water extract of Se-rich yeast using preparative size exclusion, anion exchange, and capillary reverse phase columns coupled to IM-MS. 4'-nitrobenzo-15-crown-5 ether, a selective shift reagent (SSR), was added after the last chromatographic dimension in order to specifically increase the CCS of one of the isomers by the formation of a stable host-guest system with the crown ether. Both isomers were consequently fully resolved by IM-MS, and the relative ratio of the isomers was determined to be 11-13% and 87-89%. The present data compared favorably with the literature to support the analytical strategy despite the lack of an authentic standard for method validation. In addition, computational chemistry methods were successfully applied to design the SSR and to support the experimental data.