Models predictive of intestinal drug absorption are important in drug development to identify compounds with promising biopharmaceutical properties. Since permeability is a factor affecting absorption, cell culture models (e.g., Caco-2, MDCK) have been developed to predict drug transport from the intestinal lumen into the bloodstream. The differences as to how the assays are performed, along with heterogeneity of the cell lines, have lead to different permeability values for the same drug. Transport and metabolic properties of cultured cells can vary due to culture conditions, seeding density, passage number, confluency, filter support, monolayer age, and stage of differentiation. During the transport experiment, cell absorption properties can change due to the composition and pH of the transport buffer, solute concentration and solubility, temperature, additives and/or cosolvents, agitation, sampling schedule, sink conditions, and analytical methods. Such variability within a laboratory can be avoided by characterizing a cell culture method and setting acceptance criteria in terms of monolayer integrity, passive transport, and active transport. The repeated evaluation of reference compounds will then facilitate intra-laboratory comparisons.