This study reports on a novel, high-throughput assay, designed to predict passive, transcellular permeability in early drug discovery. The assay is carried out in 96-well microtiterplates and measures the ability of compounds to diffuse from a donor to an acceptor compartment which are separated by a 9-10 microm hexadecane liquid layer. A set of 32 well-characterized, chemically diverse drugs was used to validate the method. The permeability values derived from the flux factors between donor and acceptor compartments show a good correlation with gastrointestinal absorption in humans. For comparison, correlations based on experimental or calculated octanol/water distribution coefficients (log D(o/w,6.8)) were significantly lower. In addition, this simple and robust assay allows determination of pH permeability profiles, critical information to predict gastrointestinal absorption of ionizable drugs and difficult to obtain from cell culture experiments. Correction for the unstirred water layer effect allows to differentiate between effective and intrinsic membrane permeability and opens up the dynamic range of the method. In addition, alkane/water partition coefficients can be derived from intrinsic membrane permeabilities, making this assay the first high-throughput method able to measure alkane/water log P in the microtiterplate format.