© 2022 Elsevier LtdIn this study, a series of the novel Tetracaine derivatives bearing hydrazide-hydrazone moiety were designed, synthesized, and evaluated for anticancer activity. The structures of these compounds were characterized by spectral (1H–13C NMR, FT-IR, and HR-MS analyses) methods. All synthesized compounds (2a-l) were screened for anticancer activity against human hepatocellular carcinoma (HepG2) and lung carcinoma (A549) cell lines. Against HepG2 and A549 cell lines, among the synthesized compounds, 4-(Butylamino)-N'-[(2,4-dichlorophenyl)methylidene]benzohydrazide (2i) demonstrated the most potent anticancer activity with IC50 values 28 and 7 μM, respectively. Possible cytotoxic effects of compounds (2a-l) on both normal human lung fibroblast (MCR-5) and normal human dermal fibroblast (HDF) cell lines were assessed. Inhibition of anti-apoptotic protein Bax and Bcl-2 was investigated in HepG2 and A549 cells treated with compound 2i using qRT-PCR. Apoptosis was also detected by Annexin V studies. The flow cytometric analysis results showed that compound 2i treatment of HepG2 and A-549 cells significantly increased apoptotic cell populations while decreasing viabilities in these carcinomas in a dose-dependent manner after 72 h of incubation.