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Backround: Measurement of cell survival as performed by the differential staining cytotoxicity (DiSC) assay allows for a comprehensive prediction of individual chemotherapy response. This assay has not reached routine clinical application because of its several limitations. In order to improve the DiSC assay, we developed a new type of chemotherapy sensitivity and resistance assay (CSRA) by combining a novel evaluation methodology with automated measurement of cell survival based on flow cytometry, termed AutoDiSC. Materials and Methods: Tumour cell lines were treated with cytostatic drugs and cultured in 96-well plates for four days at 37°C. Subsequently, propidium iodide was added and 96-well plates were analyzed using flow cytometry in order to determine the number of living cells in each well. Each component was evaluated for reproducibility and impact on assay read-out. Results: The evaluation process allowed for specification of all relevant parameters that determine assay performance. Conclusion: AutoDiSC is a new fully-automated CSRA which meets all criteria required for routine clinical use.