Topoisomerases (topo-I and topo-II) have occupied a significant role in DNA replication, transcription, and are a promising set of antitumor targets. In the present approach, a series of new 4β-amidotriazole linked podophyllotoxin derivatives (10a-i and 11a-k) were designed, synthesized by employing the click chemistry and their biological activities were evaluated. The majority of derivatives showed promising antiproliferative activity with IC values ranging from 1 to 10 μM on the six human cancer cell lines; cervical (HeLa), breast (MCF-7), prostate (DU-145), lung (A549), liver (HepG2) and colon (HT-29). Among them, some of the congeners 10b, 10g and 10i have shown remarkable cytotoxicity with IC values of, < 1 μM against the tested cancer cell lines and found to be more active than etoposide. Topoisomerase-mediated DNA relaxation assay results showed that the derivatives could efficiently inhibit the activity of topoisomerase-II. In addition, flow cytometry analysis on DU-145 cells revealed that these compounds arrest G2/M phase of cell cycle. Further apoptotic studies were also performed on these DU-145 cells, which showed that this class of compounds could induce apoptosis effectively.