Here, we described a unique simultaneous electrorotation (ROT) device for monitoring the rotation rate of Jurkat cells via chemical stimulation without fluorescent labeling and an algorithm for estimating cell rotation rates. The device comprised two pairs of interdigitated array electrodes that were stacked orthogonally through a 20 μm-thick insulating layer with rectangular microwells. Four microelectrodes (two were patterned on the bottom of the microwells and the other two on the insulating layer) were arranged on each side of the rectangular microwells. The cells, which were trapped in the microwells, underwent ROT when AC voltages were applied to the four microelectrodes to generate a rotating electric field. These microwells maintained the cells even in fluid flows. Thereafter, the ROT rates of the trapped cells were estimated and monitored during the stimulation. We demonstrated the feasibility of estimating the chemical efficiency of cells by monitoring the ROT rates of the cells. After introducing a Jurkat cell suspension into the device, the cells were subjected to ROT by applying an AC signal. Further, the rotating cells were chemically stimulated by adding an ionomycin (a calcium ionophore)-containing aliquot. The ROT rate of the ionomycin-stimulated cells decreased gradually to 90% of the initial rate after 30 s. The ROT rate was reduced by an increase in membrane capacitance. Thus, our device enabled the simultaneous chemical stimulation-induced monitoring of the alterations in the membrane capacitances of many cells without fluorescent labeling.