© 2022In the present study, we report the synthesis, cytotoxicity determination and molecular modeling studies of twelve novel indole-based hydrazone derivatives (IH1-IH12). To obtain the target molecules, initially, 1H-indole-2-carboxylic acid and ethanol were heated in the presence of an acid catalyst to yield ethyl 1H-indole-2-carboxylate. Following the hydrazinolyzation of the ester moiety, the resulting compound, 1H-indole-2-carbohydrazide reacted with appropriate benzaldehyde derivatives to obtain IH1-IH12. The proposed chemical structures of all compounds were confirmed by their 1H NMR, 13C NMR, IR, and HRMS data. Additionally, the configuration of C=N bond in IH8 was determined as (E) by applying 2D NMR technique, NOESY. Subsequently, the compounds were tested against both colon cancer (HCT116) and lung cancer (A549), as well as healthy lung fibroblast (MRC-5) cell lines to determine their potential as anticancer agents and their selectivity indexes (SI). Based on the obtained data from the antiproliferative MTT assay, HCT116 cell line was more sensitive to our molecules compared to A549. Furthermore, lipophilic halogens were preferable substituents on the phenyl ring for the selective toxicity against cancer cell lines. Drug-likeness analysis carried out by calculating important physicochemical properties of IH1-IH12 confirmed that they all obey Lipinski's rule of five. Finally, hypoxia inducible factor (HIF)-1α was suggested as the potential biological target of the compounds through molecular docking studies.