Aims: To develop a novel Vero cell assay that implements a real-time cell electronic sensing (RT-CES) system for the determination of the presence of verotoxin-producing Escherichia coli (VTEC). The assay overcomes the major drawbacks in conventional Vero cell assay, for example, labour-intensive and time-consuming. Methods and Results: Cells were grown onto the surfaces of microelectronic sensors that are integrated into the bottom surfaces of the microtiter plate. Cellular viability was monitored in real-time and quantified based on changes in the sensors electrical impedance. For cell viability measurement, the data generated on the RT-CES system correlated well with those obtained by the Vero cell assay for Verotoxins. To assess cytotoxicity, test cells growing on microelectronic sensors were treated with either supernatant from pure cultures, or stool samples. The specific neutralizing antibodies of VT1 and VT2 were used to identify specific toxins in the samples. Conclusions: The RT-CES assay provides a sensitive measurement comparable to conventional crystal violet assay. The assay has been successfully and specifically used to identify VTEC in human faecal samples. Significance and Impact of the Study: The RT-CES assay significantly shortens the testing time from 48 to 72 h required by the crystal violet assay to only 15 h with automated operation.