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Along with the rapid development of cellular biological research in recent years, there has been an urgent need for a high-speed, high-precision method of separating target cells from a highly heterogeneous cell population. Among the various cell separation technologies proposed so far, dielectrophoresis (DEP)-based approaches have shown particular promise because they are noninvasive to cells. We have developed a new DEP-based device to separate large numbers of live and dead cells of the human mammary cell line MCF10A. In this study, we validated the separation performance of this device. The results showed the successful separation of a higher percentage of cells than in previous studies, with a separation efficiency higher than 90%. In the past, there have been no confirmed cases in which a separation rate of over 90% and high-speed processing of a large number of cells were simultaneously achieved. It was shown that the proposed device can process large numbers of cells at high speed and with high accuracy., (© 2024 Wiley‐VCH GmbH.)