Holder pasteurization, a process that consists of heating the sample at 62.5 °C for 30 min, is the most widely used protocol to ensure microbial inactivation in human milk. Aiming to evaluate vitamin losses after pasteurization, three extraction methods and the subsequent chromatographic conditions were optimized. For the simultaneous determination of retinol, α-tocopherol, and β-carotene a saponification procedure was employed, while folic acid and ascorbic acid were extracted using solvent extractions, followed by high-performance liquid chromatography – diode array detection. Limits of detection were: 0.010 (retinol), 0.081 (α-tocopherol), 0.011 (β-carotene), 0.008 (folic acid), and 0.012 (ascorbic acid) (mgL−1). Trueness was verified by using a milk-based standard reference material. Concentrations in raw milk samples from a Uruguayan human milk bank were: 1.5±0.2/0.38±0.08 (retinol), 12.8±2.5/2.9±0.7 (α-tocopherol), 0.58±0.08/0.067±0.009 (β-carotene), 1.05±0.20/20.3±4.7 (folic acid), and 55.4±4.1/36.1±5.9 (ascorbic acid) (colostrum/mature, mgL−1). Statistically significant losses (p<0.05) were observed after pasteurization for all studied compounds, ranging from 6.4 % to 14.2 %. Liposoluble vitamins losses were higher in colostrum, probably due to the lower fat concentrations at this stage, while the opposite tendency was observed for hydrosoluble vitamins. Ascorbic acid was the most affected compound. This work represents the first study monitoring vitamins in donated human milk in Uruguay.