The abundance of different techniques and protocols available reflects the need for reliable in vitro methods to assess intestinal absorption of potentially bioactive compounds. Physicochemical assays try to pinpoint the molecular properties contributing to the absorption process. The end points of biologically based methods, such as cell cultures and excised tissues, account for all processes undergone by a molecule that traverses a 'living' biological membrane, a cell or tissue. On top of fundamental physical processes (e.g., solubility, diffusion) such biological methods incorporate physiological responses such as active transport and metabolism. In this review, an account of in vitro methods for the assessment of molecular properties (lipophilicity, solubility, permeability) influencing intestinal absorption is given. Their advantages and limitations and the possibilities offered by this area of research are also evaluated. The combination of results from both classes of assays (physicochemical and biological) and integration with computational models will guide future developments in this field. Finally, possible future developments including stem cell research and multiple-end point assays are discussed.