The European Commission is committed to reducing animal testing by supporting the development and regulatory uptake of New Approach Methodologies (NAMs) -mechanism-based, in vitro and in silico tools that align with the 3Rs (Replacement, Reduction, Refinement). This shift is especially important for complex chemical groups like per- and polyfluoroalkyl substances (PFAS)- a large family of persistent, bioaccumulative, and potentially toxic compounds. PFAS are widely used in products like textiles, cookware, and firefighting foams, and are now recognised globally as “forever chemicals” due to their environmental persistence and resistance to degradation.Legacy PFAS such as PFOA and PFOS are being phased out, but are often replaced by short-chain or alternative PFAS, whose toxicological profiles remain poorly understood.Traditional animal-based toxicity testing is not scalable for the 12,000+ PFAS in use- hence the urgent need for human-relevant, high-throughput testing strategies.This study aims to evaluate PFAS-induced bioenergetic disruption using: (i) Seahorse extracellular flux analysis to assess mitochondrial and glycolytic function; (ii) In vitrocytotoxicity assays for comparative analysis (CCK-8 Assay); and (iii) Integration with the Adverse Outcome Pathway (AOP) framework to support regulatory decision-making.