Carbohydrates play important roles in biological processes, but their identification remains a significant analytical problem. While mass spectrometry has increasingly enabled the elucidation of carbohydrates, current approaches are limited in their abilities to differentiate isomeric carbohydrates when these are not separated prior to tandem−mass spectrometry analysis. This analytical challenge takes on increased relevance because of the pervasive presence of isomeric carbohydrates in biological systems. Here, we demonstrate that TIMS 2 −MS 2 workflows enabled by tandem-trapped ion mobility spectrometry−mass spectrometry (tTIMS/MS) provide a general approach to differentiate isomeric, nonseparated carbohydrates. Our analysis shows that (1) cross sections measured by TIMS are sufficiently precise and robust for ion identification; (2) fragment ion cross sections from TIMS 2 analysis can be analytically exploited to identify carbohydrate precursors even if the precursor ions are not separated by TIMS; (3) low-abundant fragment ions can be exploited to identify carbohydrate precursors even if the precursor ions are not separated by IMS. (4) MS 2 analysis of fragment ions produced by TIMS 2 can be used to validate and/or further characterize carbohydrate structures. Taken together, our analysis underlines the opportunities that tandem-ion mobility spectrometry/MS methods offer for the characterization of mixtures of isomeric carbohydrates.