Abstract A high-performance liquid chromatographic (HPLC) method was developed for the determination, in one run, of retinol and α-tocopherol in infant milk formulae. The method involved saponification at room temperature and a later extraction of vitamins with n-hexane. The vitamins were resolved with a C 18 reversed-phase column and they were detected by UV spectrophotometry. Linearity, precision, recovery and sensitivity were satisfactory. The main advantage of the method proposed is the simultaneous determination of both vitamins using a common extraction procedure and UV detection with a variable-wavelength spectrophotometer.