The efflux pump P-glycoprotein (ATP-binding cassette B1, multidrug resistance [MDR] 1, P-gp) has long been known to contribute to MDR against cancer chemotherapeutics. We describe the development of a dual-fluorescent cell line system to allow multiplexing of drug-sensitive and P-gp-mediated MDR cell lines. The parental OVCAR-8 human ovarian carcinoma cell line and the isogenic MDR NCI/ADR-RES subline, which stably expresses high levels of endogenous P-gp, were transfected to express the fluorescent proteins Discosoma sp. red fluorescent protein DsRed2 and enhanced green fluorescent protein, respectively. Co-culture conditions were defined, and fluorescent barcoding of each cell line allowed for the direct, simultaneous comparison of resistance to cytotoxic compounds in sensitive and MDR cell lines. We show that this assay system retains the phenotypes of the original lines and is suitable for multiplexing using confocal microscopy, flow cytometry, or laser scanning microplate cytometry in 1,536-well plates, enabling the high-throughput screening of large chemical libraries.