Endothelial cells line the inner surface of all blood and lymphatic vessels, creating a semi-permeable barrier regulating fluid and solute exchange between blood or lymph and their surrounding tissues. The ability of a virus to cross the endothelial barrier is an important mechanism that facilitates virus dissemination in the human body. Many viruses are reported to alter endothelial permeability and/or cause endothelial cell barrier disruption during infection, which is able to cause vascular leakage. The current study describes a real-time cell analysis (RTCA) protocol, using a commercial real-time cell analyzer to monitor endothelial integrity and permeability changes during Zika virus (ZIKV) infection of the human umbilical vein endothelial cells (HUVECs). The impedance signals recorded before and after ZIKV infection were translated to cell index (CI) values and analyzed. The RTCA protocol allows the detection of transient effects in the form of cell morphological changes during a viral infection. This assay could also be useful for studying changes in the vascular integrity of HUVECs in other experimental setups.