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Onsite determination of cell concentration in 3D cell culture construct is proposed based on electrical impedance measurement through on-chip vertical electrodes embedded in the 3D cell culture chamber. Cells were encapsulated in agarose gel and cultured in the 3D cell culture chamber. A pair of vertical electrodes was fabricated at the opposite sidewalls of the culture chamber for performing electrical impedance measurement. Electric field penetrated through the cells/agarose construct and the cell concentration in the construct can be estimated by the impedance change. In this study, cell concentrations of 104, 105, and 106 cells/ml can be determined directly without altering the cell property.