The authors describe an assay to quantitate DNA fragmentation using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) stain, adapted to a 96-well microplate format for adherent cells, and an automated high-content screening imager. The apoptotic responses to actinomycin D (a known antineoplastic agent) to imiquimod (a small-molecule toll-like receptor [TLR] 7 agonist used in skin cancer treatment) and to several structurally related TLR 7/8 agonists were evaluated in squamous carcinoma SCC15 and SCC25 cells and normal human keratinocytes. Potent proapoptotic and growth-impairing (as determined by reduced cell numbers) actions of actinomycin D (1-300 ng/mL) were discerned with the assay. Consistent with previous reports, imiquimod (at 300 microM; approximately 75 microg/mL) induced TUNEL positivity of malignant cell cultures, but this effect also occurred in normal keratinocytes. Two related TLR agonists induced apoptosis at lower concentrations. However, the concentrations of these and the imiquimod necessary to elicit cancer cell apoptosis were 300 to 1000 times higher relative to their ability to induce the secretion of an antineoplastic protein, interferon-alpha, from human blood monocytes. This TUNEL analysis allows the quantitative comparison of compounds' apoptotic activity toward adherent malignant and normal cells and may be useful for hit characterization after a screen.