A microelectronic sensor-based platform, the RT-CES (real time electronic sensing) system, is introduced for label free assessment of natural killer (NK) cell-mediated cytotoxic activity. The RT-CES system was used to dynamically and quantitatively monitor NK-mediated cytotoxic activity towards 8 different adherent target cell lines, including cancer cell lines commonly used in laboratories. The cytotoxic activity monitored by RT-CES system was compared with standard techniques such as MTT measurement and shows good correlation and sensitivity. To test the specificity of the assay, pharmacological agents that inhibit NK cell degranulation and cytotoxic activity were employed and were shown to selectively and dose-dependently inhibit NK-mediated cytotoxic activity toward target cells. In summary, the RT-CES system offers fully automated measurement of cytotoxic activity in real time, which enables large-scale screening of chemical compounds or genes responsible for the regulation of NK-mediated cytotoxic activity.