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In oncology, diagnostic assays have the potential to individualize treatment. Due to a large number of chemotherapeutic agents available, a chemosensitivity assay would be of great value for patients receiving chemotherapy. However, no broadly accepted test exists to date. Our work aims at establishing a chemosensitivity test using a microphysiometer, a device that measures the extracellular acidification rate of cells. The test assesses chemosensitivity by comparing the acidification rate of cells treated with cytostatic agents with that of nontreated cells. We performed experiments with two human colon tumor cell lines, HCT 116 and SW620, and cytostatic agents commonly used in therapy of colon carcinoma. We showed that a chemosensitivity test can be reproducibly performed using a microphysiometer. Suitable media and conditions for the assay were found. Test results were further correlated with the crystal violet assay and a tetrazolium salt assay. In comparison, cytostatic effects were rapidly and most clearly displayed with the microphysiometer. Our results suggest that microphysiometry is a promising platform for chemosensitivity testing.