Per- and polyfluoroalkyl substances (PFAS) are a large group of anthropogenic chemicals that have been applied in industrial, commercial, and domestic products since the 1950s. Because of their toxicity, persistence, and bioaccumulation potential, PFAS have become global environmental pollutants. Besides the environment, the food chain represents another source of exposure, and the risk related to the presence of PFAS in foods has become of increased interest. Whole milk, infant formula, and ingredients used in infant formula production represent important foodstuffs that require sensitive methods with reporting limits at low parts per billion levels or lower for multiple PFAS. This article summarizes optimization experiments and validation of a complete workflow, including sample preparation and a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of sixteen priority PFAS analytes listed by the U.S. Food and Drug Administration (FDA).