We report a real-time cell analysis (RTCA) sensing method of 96 electronic microwells for profiling the cytotoxicity of nanoparticles on different cell lines. The method consists of 96 microwells embedded with microelectrodes (96x E-plate) to measure impedance changes of adherent cell lines. When the testing cells change in population, adhesion, and/or morphology, the impedance at the cell-electrode interface changes to provide real-time monitoring of overall cell status. To demonstrate this technique, we used three cell lines as sensing probes: two human lung carcinoma cell lines, A549 and SK-MES-1, and a normal mammalian cell line, CHO-K1. We tested two well-characterized nanoparticles: nano-titanium dioxide (nTiO2) and nano-silver (nAg). The three cell lines were separately seeded into 96x E-plates and treated with varying concentrations of nanoparticles (0.078-160 μg mL(-1)). This method provides dynamic cell response profiles and temporal IC50 histograms, showing concentration-, time-, particle-, and cell-dependent cytotoxicity. The 24 h and 48 h IC50 values of nAg obtained using both the RTCA and the neutral red uptake (NRU) assays were in good agreement, validating the RTCA technique. The RTCA assay does not suffer interference from nTiO2, whereas the NRU assay cannot be used due to severe interference from nTiO2. A cytostatic response was observed in CHO-K1 cells after 24 h exposure to 40 μg mL(-1) nTiO2, which was correlated with S-phase cell cycle arrest based on cell cycle analysis using flow cytometry. This suggests that the shapes of the response curves provide indicative information, directing further studies into the mode of action of the toxicant. Advantages of the RTCA technique over traditional colorimetric assays for screening the cytotoxicity of nanoparticles include minimizing interference, qualitative and quantitative cytotoxicity data, and the capability of real-time and high-throughput measurements.