In recent years, production of biosynthesized human milk oligosaccharides (HMOs) has become scalable to industrial standards. As a result, infant formula fortified with 2'-fucosyllactose (2'-FL), the most abundant HMO in human breast milk, is now commercially available. 2'-FL and 3-fucosyllactose (3-FL), another abundant HMO, are thought to be beneficial for infant health and development. Products containing HMOs are projected to expand in the future, showing the need for robust, easily applicable analytical methods for the quantitative assessment of HMOs in different food applications. We present here a validated high-performance liquid chromatography method for the quantification of 2'-FL and 3-FL in whole milk, infant formula, and cereal bars. The sample preparation was simple dispersion and extraction of the sample. The samples were analyzed by hydrophilic interaction liquid chromatography with refractive index detection and a runtime of 19 min. The method had a high degree of linearity (R 2 > 0.9995) in the range 0.2 to 12 mg/mL. The recovery for 2'-FL was 88% to 105% and for 3-FL 94% to 112%. The limit of detection (LOD) for whole milk was 0.1 mg/mL for 2'-FL and 0.2 mg/mL for 3-FL. In infant formula and cereal bars, the LOD was 0.6 mg/g for both 2'-FL and 3-FL. To show the practical application of this method, it was successfully utilized in stability studies of 2'-FL and 3-FL in whole milk, UHT milk, and yoghurt. The method provides a means of simultaneous and robust quantification of 2'-FL and 3-FL in various food matrices with high accuracy and high reproducibility.Practical Application: 2'-Fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) are two of the most abundant human milk oligosaccharides (HMOs) present in human breast milk. We present a fast HPLC method for the robust quantification of these two compounds in infant formula, whole milk, UHT milk, cereal bars, and yoghurt. This method can easily be set up by food producers and researchers to analyze the dosage of 2'-FL and 3-FL in their product or perform shelf life studies in different food applications.