Permeability is a key factor driving the absorption of orally administered drugs. In early discovery, the efficient evaluation of permeability, particularly for compounds violating Lipinski's Rule of 5, remains challenging. Addressing this, we established a high-throughput method to measure the experimental polar surface area (HT-EPSA) as an in vitro surrogate to measure permeability. Compared to earlier methods, HT-EPSA significantly reduces data acquisition time with enhanced sensitivity, selectivity, and data quality. In the effort of translating EPSA to human in vitro and in vivo passive permeability, we demonstrated the application of EPSA for predicting Caco-2 cell and human intestinal permeability, showing improvements over topological polar surface area and the parallel artificial membrane permeability assay for rank-ordering permeability in a proteolysis targeting chimera case study. The HT-EPSA method is expected to be highly beneficial in guiding early stage compound rank-ordering, faster decision-making, and in predicting in vitro and/or in vivo human intestinal permeability.