Epidemiological data have shown a link between dietary intake of tomatoes and tomato products (rich in carotenoids) and a decreased risk of chronic diseases. The carotenoid profile in tomato products depends on tomato variety as well as the thermal conditions used in processing. The final carotenoid profile may affect the bioaccessibility and bioavailability of these biomolecules. Therefore, nondestructive, reliable methods are needed to characterize the structural and stereochemical variation of carotenoids. CDCl(3) rapid extraction was used to extract carotenoids from tomato juice as an alternative rapid procedure that minimizes solvents and time consumption prior to NMR analysis. The profile of these biomolecules was characterized by application of high-resolution multidimensional NMR techniques using a cryogenic probe. The combination of homonuclear and heteronuclear two-dimensional NMR techniques served to identify (all-E)-, (5Z)-, (9Z)-, and (13Z)-lycopene isomers and other carotenoids such as (all-E)-beta-carotene and (15Z)-phytoene dissolved in the extracted lipid mixture. The use of one-dimensional NMR enabled the rapid identification of lycopene isomers, thereby minimizing further isomerization of (all-E)-lycopene as compared to HPLC data. On the basis of the assignments accomplished, the carotenoid profile of typical tomato juice was successfully determined with minimal purification procedures.