Despite the development of laboratory automation, the time and costs for carrying out various types of toxicity assays with each compound of interest remain as a burden. Thus, it limits the extent of an investigation. We have recently developed a high-content mass spectrometric (MS) assay for untargeted in vitro toxicity. In the MS assay, high resolution mass spectrometry is used directly to acquire a unique spectral pattern from living cells that have been exposed to a compound of interest. Each spectral pattern includes multiple signals of different molecular ions and their signal intensity, which combine together corresponds to a specific cellular response from a cell culture that has been exposed to the compound of interest. By comparing the spectral patterns, cellular samples with or without any treatment could be differentiated. Also, cellular samples that had been treated with different chemicals or dosage levels could be distinguished from each other.