Carotenoid composition of breast-milk samples is a significant subject in the field of infant nutrition considering, not only their contribution to the vitamin A stores but also several other health benefits achieved through mechanisms not related with the metabolism of retinoids. These pigments are exclusive of dietary origin, so that their distribution in breast milk is a precise biomarker of quality of dietary habits correlated with the assimilation of other food lipids and the subsequent processing during lactogenesis. Accurate qualitative and quantitative determination of the carotenoid profiles in breast-milk samples is described in the present protocol, with the application of HPLC method hyphenated with mass spectrometry. The protocol provides the user with information and tools to learn and understand the behavior of these pigments under MS analysis, with the application of postprocessing steps to raw data. A particular emphasis is given to xanthophyll esters that feature colostrum secretion and picture the carotenoid profile during transition to mature milk.