Ex vivo drug sensitivity testing is of considerable benefit in aiding the choice of optimum chemotherapy for leukaemia patients, especially when several therapeutic options exist, e.g. for relapsed chronic lymphocytic leukaemia (CLL). We have used the Differential Staining Cytotoxicity (DiSC) assay to assess drug sensitivity in CLL for over a decade and here present many methodological improvements, including depositing multiple samples per microscope slide and performing a rapid LC90 evaluation. Using these improvements, 412/450 specimens were successfully tested. Failures were mainly due to extended specimen transit time. All 38 drugs tested exhibited dose-dependent cell kill and broad ranges of resultant LC90S were observed. Comparison of 2- and 4-day incubations underscored a requirement for 4-day incubation with pentostatin and steroids. The rapid, simple and streamlined DiSC assay presented here can aid choice of optimum therapy, identify novel anticancer agents and be used to study drug resistance.