Toxicity testing, including testing for skin toxicity, is essential for certification of novel pharmaceutical, chemical, and skincare products. The in vitro assessment models are considered to be the most promising; a number of such tests have been introduced into practice of approval testing. The new possibilities of detecting the early cellular response to damage can be provided by the cell-based sensors built upon visual quantification of the changes in activity of the signaling pathways involved in realization of such response. NF-κB and AP-1 represent two important protein transcription factors, the increase in activity of which in the cell is associated with damage, inflammation or redox balance alteration. The study was aimed to develop the cell-based sensors built upon the HaCaT immortalized human keratinocyte cell line that express green fluorescent protein (GFP) when the NF-κB (HaCaT/NF-κB) or AP-1 (HaCaT/AP-1) signaling pathway is activated, as well as to assess their information capacity when recording the dose-dependent response to the exposure to inducers of appropriate signaling pathways. The findings showed that the HaCaT/NF-κB cell fluorescence levels changed by 6.05 ± 0.51 and 5.53 ± 0.52 times upon exposure to TNFα or LPS (at a concentration of 0–80 ng/mL) in a dose dependent manner. The HaCaT/AP-1 biosensor also responded to the exposure to Cd (NO₃)₂ (at a concentration of 0–40 µМ) and ultraviolet A (UVA) (0–40 J/cm²), however, it enabled qualitative, but not quantitative detection. The censor cell fluorescence increased by 1.51 ± 0.24 and 1.66 ± 0.43 times, respectively. The cell-based sensors developed can be used to assess cytotoxic effects of the test substances on the human skin cells in vitro and study the cytotoxicity mechanisms.