The principle of drug sensitivity testing is to expose cancer cells to a library of different drugs and measure its effects on cell
viability. Recent technological advances, continuous approval of targeted therapies, and improved cell culture protocols have
enhanced the precision and clinical relevance of such screens. Indeed, drug sensitivity testing has proven diagnostically valuable for
patients with advanced hematologic cancers. However, different cell types behave differently in culture and therefore require
optimized drug screening protocols to ensure that their ex vivo drug sensitivity accurately reflects in vivo drug responses. For
example, primary chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) cells require unique microenvironmental stimuli
to survive in culture, while this is less the case for acute myeloid leukemia (AML) cells. Here, we present our optimized and validated
protocols for culturing and drug screening of primary cells from AML, CLL, and MM patients, and a generic protocol for cell line
models. We also discuss drug library designs, reproducibility, and quality controls. We envision that these protocols may serve as
community guidelines for the use and interpretation of assays to monitor drug sensitivity in hematologic cancers and thus
contribute to standardization. The read-outs may provide insight into tumor biology, identify or confirm treatment resistance and
sensitivity in real time, and ultimately guide clinical decision-making.