·A new series of new thiadiazol derivatives has been designed and synthesized as VEGFR-2 inhibitors.·VEGFR-2 inhibitory activity and cytotoxic effect were assessed.·The effect on cell cycle and apoptosis was determined.·In silico docking, MD simulation, ADMET, and toxicity studies were carried out. The presented study reports the synthesis, in silico and in vitro evaluations of a series of novel thiadiazole derivatives designed with the pharmacophoric features of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) inhibitors. A comprehensive assessment against MCF-7 and HepG2 cancer cell lines revealed promising inhibitors, particularly, N-(4-((E)-1-(((Z)-5-acetyl-3-(2-chlorophenyl)-1,3,4-thiadiazol-2(3H)-ylidene) hydrazono) ethyl) phenyl)-4-chlorobenzamide (compound 22) exhibited a significantly low half-maximal inhibitory concentration (IC50) value against both MCF-7 and HepG2 cells of 0.03 µM and 0.39 µM, respectively. The rest of compounds exhibited also significant IC50 values against MCF-7 and HepG2 cells of (0.07–4.7 µM) and (0.12–8.75 µM), respectively. Furthermore, compound 22 displayed a VEGFR-2 inhibitory activity (VEGFR-2 = 0.042 µM) comparable to the established inhibitor sorafenib (VEGFR-2 = 0.041 µM). Detailed cell cycle analysis demonstrated that compound 22 induced a G0-G1 phase arrest of the MCF-7 cells (71.33 %), indicative of its potential to modulate cell cycle progression. Notably, compound 22 also exhibited a substantial increase in apoptosis in the MCF-7 cells, particularly in early (22.04 %) and late stages (13.94 %), underscoring its remarkable pro-apoptotic effect. Additionally, Compound 22 demonstrated a notable decrease in the migratory ability and wound healing capacity of MCF-7 cells. Computational studies, including molecular docking, revealed high binding affinities. Over a 200 ns MD simulation, the VEGFR-2-compound 22 complex initially displayed a rapid increase in RMSD followed by stability around 3.5 Å due to ligand conformational change while maintaining stable protein binding. This was confirmed by RMSF, SASA, RoG, and hydrogen bonding analysis. MM-GBSA, ProLIF, PCAT and FEL studies confirmed VEGFR-2-compound 22 complex binding at both dynamic and energetic levels. The computational ADMET studies demonstrated favorable pharmacokinetic properties. Furthermore, Density Functional Theory (DFT) studies confirmed the reactivity and stability of compound 22. These comprehensive findings underscore the potential of compound 22 as a lead candidate for further investigation in the development of VEGFR-2-targeted anticancer agents. Display omitted