The identification of compounds that specifically inhibit or kill cancer cells without affecting cells from healthy tissues is very challenging but very important for reducing the side effects of current cancer therapies. Hence, there is an urgent need for improved assays allowing the selectivity of a given compound to be monitored directly. The authors present an assay system based on the competitive co-cultivation of an excess of cancer cells with a small fraction of noncancer human indicator cells generating a fluorescence signal. In the absence of a specific anticancer compound, the cancer cells outgrow the indicator cells and abolish the fluorescence signal. In contrast, the presence of specific anticancer drugs (such as Tyrphostin-AG1478 or PLX4720) results in the selective growth of the indicator cells, giving rise to a strong fluorescence signal. Furthermore, the authors show that the nonspecific cytotoxic compound sodium azide kills both cancer and noncancer cells, and no fluorescence signal is obtained. Hence, this assay system favors the selection of compounds that specifically target cancer cells and decreases the probability of selecting nonspecific cytotoxic molecules. Z factors of up to 0.85 were obtained, indicating an excellent assay that can be used for high-throughput screening.