A series of new indeno[1,2-b]quinoxalin derivatives were synthesized as a part of our aim to develop promising anticancer agents. In vitro anticancer effect of twenty-three of the synthesized compounds were investigated in three human cancer cell lines; the colon cancer cell line HCT-116, the liver cancer cell line HepG-2, and the breast cancer cell line MCF-7. Among the tested derivatives, 2a, 2b, 2d, 8b, 8c and 10d showed exceptional anti-proliferative activities against the three tested cell lines compared to the reference standard Imatinib. That's why, they were selected for further investigations. Evaluation of their cytotoxicity against normal human cell line (WI-38) was performed, to ensure their safety and selectivity (IC50 >92 μM). Then, induction of apoptosis by the most active compounds was found to be accomplished by down-regulation of Bcl-2 and up-regulation of BAX and Caspase-3. After that, the most promising apoptotic compound that increase the Caspase 3 and BAX, and down regulate Bcl-2 activity (2b), was assessed for the impact on the cell cycle distribution in HepG-2 cells. The investigations have shown that the most potent derivative 2b, induced cell cycle arrest at G2/M phase. Finally, in silico evaluation of ADME properties indicated that compound 2b is orally bioavailable and can be readily synthesized on a large scale.