The ATP-based tumor chemosensitivity assay (ATP–TCA) is a standardised system which can be adapted to a variety of uses with both cell lines and primary cell cultures. It has a strong track record in drug development, mechanistic studies of chemoresistance and as an aid to clinical decision-making. The method starts with the extraction of cells in suspension from continuous cell culture (for cell lines), malignant effusions or biopsy material. Enzymatic digestion is used to obtain cells from tumor tissue. The assay uses a serum-free medium and polypropylene plates to prevent the growth of non-neoplastic cells over a 6-day incubation period followed by detergent-based extraction of cellular ATP for measurement by luciferin–luciferase assay in a luminometer. The assay results are usually shown as percentage inhibition at each concentration tested, and can be used with suitable software to examine synergy between different anticancer agents.