In this study, we report the use of a real-time cell analysis (RTCA) test system, the xCELLigence(®) RTCA, as efficient tool for a fast cytotoxicity analysis and comparison of four different vertebrate cell cultures. This new dynamic real-time monitoring and impedance-based assay allows for a combined measurement of cell adhesion, spreading and proliferation. Cell cultures were obtained from mouse, rat, human and fish, all displaying a fibroblast-like phenotype. The measured impedance values could be correlated to characteristic cell culture behaviours. In parallel, relative cytotoxicity of a commonly used but due to its very good water solubility highly hazardous pesticide, copper sulfate, was evaluated under in vitro conditions through measurements of cell viability by classical end-point based assays MTT and PrestoBlue(®). Cell line responses in terms of viability as measured by these three methods were variable between the fish skin cells and cells from higher vertebrates and also between the three methods. The advantage of impedance-based measurements is mainly based on the continuous monitoring of cell responses for a broad range of different cells, including fish cells.