Purpose: The adenosine-triphosphate-based chemotherapy response assay (ATP-CRA) is a well-documented and validated technology for individualizing chemotherapy in cancer patients. We evaluate the feasibility of ATP-CRA in colorectal cancer patients. This study will illustrate the assay's success rate, the mean coefficient of variation, and the turnaround time as a validation tool for a chemosensitivity test. Methods: A total of 118 patients, treated by surgery between June 2004 and September 2005 were evaluated for chemosensitivity to seven anticancer agents (5-fluorouracil (5-FU), oxaliplatin, irinotecan, epirubicin, etoposide, gemcitabine, and paclitaxel) by using an ATP-CRA. To allow a comparison between samples, we calculated the chemosensitivity index (CI) based on the percentage cell death rate (CDR, %) at each test drug concentration. Results: The assay success rate was 85.5% (118/138), and the mean coefficient of variation, indicating intra-assay error level, was 9.2%. CDR measured at a therapeutic peak plasma concentration ranged from 0% to 93.6% with a median of 31.0% for 5-FU, 28.5% for oxaliplatin, 34.0% for irinotecan, 25.0% for epirubicin, 21.0% for etoposide, 22.0% for gemcitabine, and 25.2% for paclitaxel. According to the CI, the most sensitive drug varied from patient to patients 10.9% for 5-FU, 10.9% for oxaliplatin, 24.7% for irinotecan, 11.8% for epirubicin, 22.4% for etoposide, 1.1% for gemcitabine, and 23.3% for paclitaxel. Conclusions: Our data suggest that the ATP- CRA is a feasible in-vitro chemosensitivity test in colorectal cancer and that patients show heterogeneous chemosensitivity. A study evaluating the predictive value of ATP-CRA directed therapy is needed to determine the clinical usefulness of the test.