The objective of this study was to develop a method to simultaneously quantify vitamins A and E in infant formula. To determine the vitamin A and E content, vitamin A and four different vitamin E isomers (α-, β-, γ-, and δ-tocopherol) were separated by high performance liquid chromatography with a photodiode array detector using a Develosil RPAQUEOUS RP-C 3 0 column (4.6×250 mm, 5 µm). The vitamin A and E contents in the certified reference material determined using this method were within the certified range of standard values. The limits of detection (LODs) and limits of quantitation (LOQs) for vitamin A were 0.02 and 0.06 µg/L, respectively. LODs and LOQs for the vitamin E isomers ranged from 0.20 to 0.55 and from 0.67 to 1.81 µg/L, respectively. Linear analyses indicated that the square of the correlation coefficient for the vitamin A and E isomers was 0.9997-0.9999. The recovery of vitamins ranged from 96.69 to 97.79%. The results demonstrate that this novel method could be used to reliably analyze vitamin A and E content in infant formula.