We describe here the development and implementation of a pilot-scale, in vitro, anticancer drug screen utilizing a panel of 60 human tumor cell lines organized into subpanels representing leukemia, melanoma, and cancers of the lung, colon, kidney, ovary, and central nervous system. The ultimate goal of this disease-oriented screen is to facilitate the discovery of new compounds with potential cell line-specific and/or subpanel-specific antitumor activity. In the current screening protocol, each cell line is inoculated onto microtiter plates, then preincubated for 24–28 hours. Subsequently, test agents are added in five 10-fold dilutions and the culture is incubated for an additional 48 hours. For each test agent, a dose-response profile is generated. End-point determinations of the cell viability or cell growth are performed by in situ fixation of cells, followed by staining with a protein-binding dye, sulforhodamine B (SRB). The SRB binds to the basic amino acids of cellular macromolecules; the solubilized stain is measured spectrophotometrically to determine relative cell growth or viability in treated and untreated cells. Following the pilot screening studies, a screening rate of 400 compounds per week has been consistently achieved.