Carotenoids are natural pigments with provitamin A and antioxidant activities. Biosynthesized in plants as their all-trans isomers, carotenoids isomerize in solution and in humans to multiple cis isomers which can have different bioavailabilities and functions. Since separation and characterization of isomeric carotenoids using high-pressure liquid chromatography (HPLC) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) is time-consuming, the potential for ion mobility mass spectrometry (IM-MS) to resolve and characterize carotenoid isomers rapidly without chromatography was investigated using traveling-wave ion mobility spectrometry on a quadrupole time-of-flight mass spectrometer. The all-trans isomers of lycopene and β-carotene were separated by several milliseconds from the cis-isomers which were detected as partially overlapping peaks. The collision cross-section values of these carotenoid isomers were determined using IM-MS to be 180 and 236 Å(2) for cis-lycopene and all-trans-lycopene, and 181 and 225 Å(2) for cis-β-carotene and all-trans-β-carotene, respectively. Collision-induced dissociation MS/MS of ion mobility-resolved isomers indicated that cis and all-trans carotenoid isomers can be distinguished by their fragmentation patterns. Previous MS/MS studies of cis- and all trans-carotenoids had suggested that they produced identical tandem mass spectra, but this appears to have been the result of isomerization during ionization. Introduction of specific cis or trans isomers by infusion or HPLC resulted in cis/trans isomerization in the ion source during electrospray, and the relative levels of cis carotenoids forming in the ion source compared to the all-trans isomers were temperature dependent.