In this work, a non-invasive measurement technique for the quantitative determination of cell viability in a three-dimensional (3D) cell culture construct is proposed. This technique is based on on-site electrical impedance measurement. A microfluidic chip with a 3D culture chamber is fabricated to demonstrate this technique. In vitro 3D cell culture has been interpreted for faithfully representation of the in vivo cellular responses in 3D cell culture construct is normally time-consuming and labor-intensive. In this study, the microfluidic chip consists of a culture chamber, in which a pair of vertical electrodes at its opposite sidewalls was embedded, and a fluidic channel for drug perfusion. Cancer cells encapsulated in agarose gel were loaded into the culture chamber to perform 3D cell culture under the perfusion of culture medium and anti-cancer drug in different concentrations (6, 12, 18, and 24 µg/ml) for 2 days. Since higher drug concentration led to more cell damage or death, the total impedance magnitude of the culture construct was shown to be reasonably proportional to the anti-cancer drug concentration. Moreover, cell proliferation can be also monitored using this technique. The proposed measurement method can determine cell viability without affecting the cellular behaviors during culture. It has a high potential to develop a fast and easy measurement compared with the conventional cellular analysis techniques.