The MDR 1-encoded P-glycoprotein (Pgp), when overexpressed in tumor cells, confers resistance to many clinically important classes of anticancer drugs. This phenomenon is called multidrug resistance (MDR). The finding that this gene was expressed in many types of human cancers has stimulated many studies into the relevance of this protein for clinical chemotherapy resistance (1 ). Pgp is a protein that causes a net transport of substrate drug molecules over the plasma membrane of the cell, resulting in a lowered free cytosolic drug concentration. Therefore, the drug target(s) “feel” a lower drug concentration, resulting in less drug-induced damage and cell-kill. The measurement of the active (adenosine triphosphate [ATP]-dependent) drug transport or efflux function of Pgp is, therefore, a theoretically elegant way to quantify the number of active or “functional” Pgp molecules per cell (2 ,3 ). Such assays are called functional assays in this chapter. One disadvantage is, however, that without additional data no unambiguous evidence of the molecular nature of the transport protein is obtained, the advantage is that the relevant biological feature is measured. The latter cannot be derived easily from the mRNA or protein expression levels, because a number of factors may influence the net effect of transporter proteins, such as other membrane properties specific to certain cells (3 ,4 ). In practice, important criteria for the usefulness of any MDR assay are its specificity, sensitivity, and reproducibility.