The differential staining cytotoxicity (DiSC) assay involves in vitro drug panel testing against patient tumor cells to identify optimal therapy. This observational study investigated whether DiSC assay guided treatment could improve outcome in patients with chronic lymphocytic leukemia. A cohort of 178 patients were categorized either as sensitive to drugs in vitro and receiving a sensitive drug in vivo, sensitive in vitro but not treated with a sensitive drug, or having disease resistant to all drugs tested in vitro. Response and survival for these patient categories were compared using multivariate regression techniques. Patients receiving a sensitive drug, compared with those who though having sensitivity did not, had a higher remission rate (odds ratio, 6.5; 95% CI, 2.91–14.53) and reduced death rate (hazard ratio, 0.29; 95% CI, 0.16–0.53). Having adjusted for all known confounding factors, the results suggest that in vitro drug sensitivity is an important independent prognostic variable to include in future trials, and that the DiSC assay may be a cost-effective use of health resources: the estimated incremental cost-effectiveness was £1,470 per life-year gained. A randomized controlled trial is required to confirm the benefit and estimate reliably the potential impact of assay-guided choice of therapy.