Cigarette smoke contains abundant toxicants, and profiling its cytotoxicity represents a critical topic. In this study, cell-substrate impedance sensing (ECIS) was used to measure the cytotoxicity of 4-(methylnitrosoamino)-1-(3-pyridinyl)-1-butanone (NNK), nicotine, and the total particle material (TPM) of high-tar and low-tar cigarettes on CHO-Kl cells. Normalized impedance values at 3174 Hz were collected with microscopic imaging as an assistant, showing the dynamics of cell damage and the ability for cell self-recovery. The NI determination of the four toxicants indicated that, as the concentration of toxicants increased, cigarette smoke produced more intense toxic effects on the cells, and the ability of cell self-recovery worsened until there was permanent damage to the cells and the cells eventually died. Furthermore, the survival rate of the cells was obtained during treatment. NRU assays as a comparison were developed for evaluating the cytotoxicity by calculating the IC50. Both methods showed that the cytotoxicity decreased in the following order: the TPM of high-tar cigarettes, TPM of low-tar cigarettes, nicotine and NNK. The sensitivity of the ECIS method was higher. Our work provides a useful and convenient approach for determining the cytotoxicity of cigarettes in a real-time, label-free manner, contributing to the development of low-toxicity cigarettes.