Aqueous film-forming foams (AFFFs) are complex product mixtures that often contain per- and polyfluorinated alkyl substances (PFAS) to enhance fire suppression and protect firefighters. However, PFAS have been associated with a range of adverse health effects (e.g., liver and thyroid disease and cancer), and innovative approach methods to better understand their toxicity potential and identify safer alternatives are needed. In this study, we investigated a set of 30 substances (e.g., AFFF, PFAS, and clinical drugs) using differentiated cultures of human hepatocytes (HepaRG, 2D), high-throughput transcriptomics, deep learning of cell morphology images, and liver enzyme leakage assays with benchmark dose analysis to (1) predict the potency ranges for human liver injury, (2) delineate gene- and pathway-level transcriptomic points-of-departure for molecular hazard characterization and prioritization, (3) characterize human hepatocellular response similarities to inform regulatory read-across efforts, and (4) introduce an innovative approach to translate mechanistic hepatocellular response data to predict the potency ranges for PFAS-induced hepatomegaly in vivo. Collectively, these data fill important mechanistic knowledge gaps with PFAS/AFFF and represent a scalable platform to address the thousands of PFAS in commerce for greener chemistries and next-generation risk assessments.