Although biological activity is a key issue in developing a potent drug, other factors such as solubility, absorption, partitioning, or biodegradation are equally important, with their optimization often situated late in the drug discovery/development process. The gastrointestinal absorption of an orally administered drug is one of the key factors for its bioavailability, but existing methods like Caco-2 cells (laboratory intensive, not suited for high-throughput measurements) and immobilized artificial membrane (IAM) columns (throughput limited by HPLC) are not ideal for early-stage high-throughput screening (HTS). Here we describe the Parallel Artificial Membrane Permeation Assay (PAMPA), a simple method for predicting transcellular drug absorption. PAMPA is based on 96-well microtiter plate technology, using a membrane formed by lecithin and an inert organic solvent on a hydrophobic filter, with no active transporter systems. It allows measuring hundreds of compounds a day via simple UV spectroscopy (parallel measurements at multiple wavelengths) for concentration analysis. Validated with a diverse set of well-described drugs (including known passively absorbed, actively transported, and paracellularly absorbed compounds), PAMPA flux values were compared with known human absorption data. The main objective of PAMPA is to classify passively transported compounds, focusing on the transcellular absorption route, and it can distinguish compounds absorbed via active transport or the paracellular route.