Per- and polyfluoroalkyl substances (PFAS) are anthropogenic compounds present in a wide range of consumer products. These chemicals have been identified as Contaminants of Emerging Concern (CECs) because they are both environmentally toxic and persistent, as well as bioaccumulative in living organisms. PFAS do not accumulate in fatty tissue but rather in predominantly protein-dense compartments, such as the liver, kidneys, and blood due to their oleophobic properties. Therefore, PFAS-protein binding studies are important in assessing their bioaccumulative potential. Human serum albumin (HSA) is the most abundant transporter protein in the human body and has been reported as a major binding protein of PFAS in the blood and as a target for bioaccumulation studies. The goal of this study is to determine PFAS-HSA protein-water distribution coefficients (KPW) for both legacy and alternative PFAS. A high-throughput equilibrium dialysis method is used under physiological conditions, coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), to quantify KPW values for a variety of PFAS with HSA. This approach allows us to investigate relationships between PFAS structure and HSA binding. The method has been validated by quantifying log KPW values for perfluorooctanoic acid (PFOA)-HSA binding and hexafluoropropylene oxide dimer-acid (GenX)-HSA binding: 4.17 ± 0.04 and 3.2 ± 0.1, respectively. These values are in agreement with literature values for these PFAS binding to bovine serum albumin (BSA, the highly homologous analog of HSA found in cows) using an equilibrium dialysis technique. Ongoing studies are focused on applying this high-throughput equilibrium dialysis method to determine KPW values for a variety of legacy and alternative PFAS in order to investigate their relative bioaccumulative potentials and their binding strengths to HSA.