The development of multiplexing capabilities and high-content readouts reporting individual cellular measurements enables assessment of biological variability on a single-cell basis, together with the evaluation of cell subpopulations within wells. A high-content screening multiplexed assay format allows additional information to be gained from a single assay. One such example is the ability to determine the effects of new chemical entities on different cell lines, tested in the same well. These assays, coupled with an appropriate automated cell-analysis platform, enable scalable screening of compound libraries for selectivity or toxicity. This approach can greatly increase screening efficiencies and enhance the amount of information achieved from a particular assay procedure, resulting in a significant reduction in the overall cost of a chemical compound library screen. By labeling live cells with Qtracker™ cell labeling kits and identifying cell proliferation using an Acumen Explorer® microplate cytometer, we were able to determine the differential rates of cell proliferation of the individual cell lines in the same well over time. This method can extend to multiplexing more than two cell populations and to measure drug-induced differential changes in proliferation in a single-well assay on multiple cell lines.