The use of Caco-2 cells for screening of discovery compounds for their permeability characteristics and P-glycoprotein interactions is well established and used routinely in pharmaceutical industries world-wide. The screening model involves growing cells on 12 or 24 well transwell format. In this manuscript, we report the use of Caco-2 cells grown on 96 well transwell plates for screening compounds for their potential to interact with P-gp. Bi-directionality studies were performed with known P-gp substrates such as saquinavir, indinavir, vinblastine, vincristine, verapamil, digoxin and taxol. P-gp inhibition studies were also conducted using radiolabeled digoxin as the probe. The results demonstrated that P-gp substrates had efflux ratios (Pc (B to A)/Pc (A to B)) in the 96 well format that were comparable to the ratios seen in 12 and 24 well format. Inhibition of digoxin efflux transport in presence of the test compounds (P-gp substrates) demonstrated that 96 well cells express adequate amounts of efflux transporters and perform as well as the 12 and 24 well Caco-2 cells. Thus, the 96 well Caco-2 cell set-up presents a higher throughput permeability model capable of identifying compounds that interact with P-gp and has the potential to significantly increase the efficiency of P-gp screening in early drug discovery.