A regional average intensity-based adherent cellular imaging assay has been applied to the evaluation of drug-induced cardiotoxicity. Unlike single cell imaging cytometry, which requires isolated single cells, regional average intensity-based cellular imaging does not require enzymatic treatment for the detachment and isolation of adherent cells. In addition, cellular morphological information is preserved. The regional average cellular intensity was obtained through defocusing adherent cells, applying a threshold to the defocused cellular region, selection of the cellular region, and overlapping the selected cellular region with its fluorescence cellular image. This concept was verified by the comparison of regional average fluorescence intensities and green fluorescent protein (GFP) expression percentages between isolated single and adherent human embryonic kidney (HEK)-293 cells with overexpressed GFPs. The regional average GFP adherent cellular intensity was almost identical to the average GFP cellular intensity obtained through single cell imaging cytometry. The effect of cisapride and dexamethasone on the cardiotoxicity present in human ether-à-go-go related gene (hERG)-overexpressed HEK-293 cells was successfully assessed using this approach. Proper evaluation of drug-induced cardiotoxicity is an important issue in the drug discovery process because many pharmaceuticals have been withdrawn from the market due to drug-induced cardiotoxicity. The obtained results demonstrated the unique potential of regional average intensity-based cellular imaging as an in vitro drug safety screening that can be well adapted at the in vitro stage before the in vivo animal model.