A rapid, sensitive and inexpensive method has been developed for the simultaneous determination of fat-soluble vitamins A and E in infant formula (IF) samples. The isolation of fat-soluble vitamins includes a saponification step and an extraction step with petroleum ether. The qualitative and quantitative determination of vitamins was performed by High Performance Liquid Chromatography (HPLC) coupled to Diode Array Detector (DAD). More specifically, method development involved optimization of saponification time, testing of antioxidants, and number of extractions along with the extraction time applied. The method proved to be reliable as results agreed with the sample’s labels and the permitted variation according to the EE law. The relative standard deviation decreased significantly after the optimization (%RSD 1.8 % for retinol and 3.1% for a-tocopherol).