P-Glycoprotein (P-gp) is a multidrug transporter responsible for resistance to anticancer chemotherapy and physiologically involved in absorption, distribution, and excretion of a large number of hydrophobic xenobiotics. P-gp exhibits both an adenosine triphosphatase (ATPase) activity correlated with its drug transport function and a basal ATPase activity in the absence of any drug. The authors have developed an enzymatic test based on modulation of these ATPase activities, which makes it possible to detect specific interactions between drugs and P-gp. They took into account the existence of multiple binding sites on P-gp to finalize an optimized strategy that involves the assay of the P-gp ATPase stimulated by three reference substrates (verapamil, vinblastine, and progesterone) in addition to its basal ATPase activity. This assay uses a coupled enzyme system with spectrophotometric detection for measurement on P-gp-containing native membrane vesicles. This assay may be performed on 96- or 384-well microplates and is therefore suitable for high-throughput screenings.