A method for the simultaneous determination of vitamin A (all-trans-retinol) and vitamin E (α-tocopherol) in milk using the HPLC technique with UV detection (324 nm, vitamin A) and fluorescent detection (Ex295 nm/Em350 nm, vitamin E) was validated. A reverse phase LiChroCART™ 250-4 Superspher™ 100 RP-18 column was used for chromatographic separation. A mixture of methanol and H₂O (96.5:3.5 v/v) was used as the eluent (1 mL/min). The analyses were performed after spectrophotometric standardization of standard ethanol solutions, and the results were corrected by recovery. The residual coefficients of variation for the regression equation y=ax were 4.7% (vitamin A) and 8.3% (vitamin E), with r² > 0.998 (for both vitamins). The limit of quantitation was 0.02 μg/mL and 0.3 μg/mL, repeatability and reproducibility 14% and 12.5%, and uncertainty of the method 20.7% and 18.8% for vitamins A and E, respectively. Vitamin recovery from milk was 51.6-75.1% (vitamin A) and 70.1-87.8% (vitamin E). The results of the reference material analyses (CRM 421, SRM 2383) concur with the certified reference values. The analytical method described is precise, accurate, fast and inexpensive.