This paper reports the separation of cells using a dielectrophoretic (DEP) chromatography device. The device consists of a micro channel and an array of interdigitated microelectrodes on a glass substrate. The sample cells were fed pulse-wise into the carrier flow using a micro-injector. The cells in the sample received a non-uniform electric field made with an electrode array. The direction of DEP motion is towards the higher field when the cell is more polarizable than the medium (positive DEP), while the direction is towards the lower field when the cell is less polarizable than the medium (negative DEP). Therefore, the cell separation depends on the size and dielectric characteristic. The effects of carrier flow rate, frequency, applied voltage, and sweep frequency on the retention time of the sample in the device were examined. In this study, mouse-hybridoma 3-2H3 cells and yeast cells were used as the sample cell. The analytical characteristic of the DEP chromatography device was evaluated according to the difference of retention time by the electric field. As a result, the separation in the cells in the negative DEP using the DEP chromatography was found to be effective. In addition, the effect of the sweep frequency on the difference in the retention time of the mouse hybridoma 3-2H3 cells and the yeast cells was very large. Consequently, the effectiveness of the DEP chromatography device was proven.