A series of novel 4β-[(5-substituted)-1,2,3,4-tetrazolyl] podophyllotoxin derivatives were synthesized by employing azide-nitrile click chemistry approach. All the derivatives were evaluated for their cytotoxicity against a panel of four human cancer cell lines and their IC50 values were found to be in the range of 2.4-29.06 μM. The cytotoxicity exhibited by the majority of test compounds were found to comparable and often more effective than doxorubicin and all compounds exhibited higher cytotoxicity on A-549 cell lines. Cell cycle analysis showed that the novel 4β-[(5-substituted)-1,2,3,4-tetrazolyl] podophyllotoxins resulted in cell cycle arrest at G2/M phase and were also found to be the potent inhibitors of tubulin polymerization in vitro.