A fast method was developed to determine carotenoid content in transgenic maize seeds. The analysis was carried out using an ultrahigh-pressure liquid chromatograph coupled to a photodiode array detector and a mass spectrometer (UHPLC-PDA-MS/MS). Sixteen carotenoid pigments were detected and quantified in 0.9952), (c) satisfactory recoveries for most of the pigments (between 82 and 108%), and (d) low detection (from 0.02 to 0.07 μg/mL) and quantification limits (from 0.05 to 0.20 μg/mL) (LOD and LOQ, respectively). The methodology was applied to the analysis of transgenic maize lines TM1, TM2, and TM3, expressing several carotenogenic genes. ; We thank the Comissionat per a Universitats i Recerca del DIUE de la Generalitat de Catalunya (Barcelona, Spain) and the European Social Fund (ESF) for a Ph.D. fellowship to S.M.R. This work was supported by the University of Lleida, Ministry of Science and Innovation (MICINN), Spain (BFU200761413 and CTQ2009-14699-C02-01), and an ERC Advanced Grant (BIOFORCE) to P.C.