Drug Permeation Dataset Across Diverse Chemical Space Using PermeaPad and High-Throughput Plate Reader Assays

Purpose High-throughput permeation assays are essential tools in drug discovery for identifying small molecules with favorable permeability, yet quantifying their transport kinetics across biomimetic membranes often relies on resource-intensive chromatographic methods. Here, we propose a simplified calibration-free endpoint absorbance workflow ( P One ) for rapid permeability screening in the 96-well PermeaPad ® format. Methods Using a single plate-reader measurement and avoiding compound-specific calibration curves during primary screening, we evaluated permeation of 42 chemically diverse small molecules selected to span a broad physicochemical space relevant to passive membrane diffusion. Results P One showed strong correlations with conventional calibration-based UV quantification ( P Cal , R 2 = 0.85) and with LC–MS/MS-based (R 2 = 0.82) methods. Using a pragmatic 10% P One threshold, P One and P Cal yielded concordant permeability classifications in 89% of cases, demonstrating the usefulness of the method for rapid compound screening. Furthermore, we evaluated the transferability of the P One workflow to a mucus-coupled permeation model to further mimic the intestinal barrier. Although mucus-dependent alteration in permeation was observed for some compounds, these experiments showed that mucus-derived matrix interference restricts direct optical quantification, limiting the applicability of the method to a subset of compounds with suitable spectral properties. Conclusion While the need for a chromophore structure limits the P One method, it offers a cost-effective, robust alternative for early-stage screening workflows.