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 <13 min. In addition, it was possible to obtain good resolution of both polar xanthophylls and nonpolar carotenes. The method exhibited (a) a high degree of repeatability (%RSD < 13%), (b) linear calibration curves (R2 > 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.