Quantification of protein binding of new chemical entities is an important early screening step during drug discovery and is of fundamental interest for the estimation of safety margins during drug development. In this publication, we describe the development of a new high-throughput assay for the determination of the free drug fraction in plasma (f u ). The new technique is an enhancement of the previously published erythrocytes partition method. It is based on the distribution of drugs between plasma water, plasma proteins, and solid-supported lipid membranes (Transil 1 ). The execution of protein binding studies by partitioning is dramatically simplified by substituting erythrocytes with commercially available Transil 1 beads, and makes the method particularly suitable for high-throughput studies. Eight Bayer compounds from different compound classes covering a wide range of lipophilicities (log P ¼ 1.9-5.6) and f u values (0.018-35%) were selected for validation of the assay. The results obtained by the new method and by either the erythrocytes partitioning technique or more conventional methods (ultrafiltration and equilibrium dialysis) are identical, confirming that the new method produces valid results even for drugs that are strongly bound to plasma proteins. Precision and accuracy of the data in the cases of very low and high f u values are comparable, indicating that the method is especially suited for highly lipophilic drugs that tend to adsorb to surfaces compared with other methods, like ultrafiltration or equilibrium dialysis, that may produce biased data. The method is also useful for the determination of binding parameters and the pH dependence of f u . In summary, this assay is well suited for high-throughput determination of protein binding during drug discovery and for extended protein binding studies during drug development.