Cell damage was determined by measuring the specific release of acetoxymethyl ester (Fura-2). The aim of this study was to establish quantitative, simple, sensitive, time-saving and practical toxicity screening in broad serial dose dilutions. The cytotoxicity of ethanol and isopropanol was examined in cultured cells using HeLa (human cervical cancer of the uterus) and Caco-2 cell lines (human colon cancer). The Fura-2 assay was compared with a well-established cytotoxicity assay, namely, the MTT assay. We applied a mathematical approach based on Hill's equation to obtain the relationship between Fura-2 assay and MTT assay. Fura-2 assay was found to detect subtle cell injury at a concentration as low as 0.17 or 0.18 M when HeLa cells were treated with ethanol or isopropanol, respectively. In Caco-2 cells, subtle cell damage was detected at a concentration of 0.17 or 0.24 M upon treatment with ethanol or isopropanol, respectively. However, subtle cell damage was never detected by MTT assay. Using this Fura-2 assay, minor cell damage induced by injurious compounds was detectable within 60 s. The present method appears to be more practical for HTS analysis, more time-efficient and more sensitive for detecting subtle cell damage than the MTT assay.