DIMSCAN is a semiautomatic fluorescence-based digital image microscopy system that quantifies relative total (using a DNA stain) or viable (using fluorescein diacetate [FDA]) cell numbers in tissue culture multiwell plates ranging from 6 to 384 wells per plate. DIMSCAN is a rapid and efficient tool for conducting in vitro cytotoxicity assays across a 4 log dynamic range. The specificity of detecting viable cells with FDA is achieved by using digital image processing and chemical quenching of fluorescence in nonviable cells with eosin Y. Average scan time for the most commonly used format, a 96-well plate, is 6 min. Cytotoxicity for neuroblastoma cell lines measured by DIMSCAN was found to be comparable to manual Trypan blue dye exclusion counts or colony formation in soft agar, but with a significantly wider dynamic range, which enables drug combination studies used to detect synergistic or antagonistic interactions. The linearity of DIMSCAN was validated (r2 = 0.99967 +/- 0.0003) for cells stained with FDA deposited using a fluorescence-activated cell sorter, documenting a dynamic range > 4 logs, and the ability to detect a single viable cell in a well 93% of the time. DIMSCAN has been used to demonstrate preclinical activity of cytotostatic and cytotoxic drugs and drug combinations that have subsequently shown activity in clinical trials.