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Research to find new treatments for cancer generally begins with in-vitro tests to measure the toxic effect of a proposed medical treatment on cell line cultures. In general, these tests are called cytotoxicity assays. The development of experimental protocols to conduct these tests is an attractive area for the application of experimental design techniques. This work describes the improvement of an experimental protocol for the colorimetric MTT assay, one of the most common cytotoxicity tests. The MTT assay is used in this case study to characterize the cytotoxic level of a novel immunotherapy agent on the well-known HeLa cervical cancer cell line. The original protocol related cytotoxicity to treatment concentration, treatment time and initial number of cells in the test. With the application of an experimental strategy based on the central composite design, it was possible to improve the protocol to drastically reduce the …