A rapid, highly sensitive and direct Ultra-High-Pressure Liquid Chromatographic method was developed and validated for quantifying -, +-, and -tocopherol in human colostrum and milk. Two reversedphasechromatographiccolumnsandtwodetectors(FluorescenceDetectororFDandPhotodiodeDetector Array or PDA) were used and both methods were independently validated. Two internal standards were selected according to the detector used. Recoveries ranged from 96.71% to 103.55% and the relative standard deviations for the within-day precision were below 6% (PDA) and 3% (FD). Both approaches enabled to achieve low detection limits, on the order of ng (PDA) or pg (FD). Only 300L of sample and a chromatographic run of less than 1.6min were enough to efficiently quantify the isomers in the colostrum and milk of Spanish women.