A series of new pyrido[2,3-d]pyrimidine derivatives were synthesized adopting click chemistry approach. The obtained compounds were characterized by spectral data analysis and evaluated for their anticancer activity. All the derivatives were subjected to in vitro MTT cytotoxicity screening assay against a panel of four different human cancer cell lines, colon carcinoma (HT-29), tongue squamous cell carcinoma (CAL27), human cervical cancer (HeLa) and breast (MCF-7). Compounds 8a, 8b, showed the highest cytotoxic activities with EC50 values of 25.21±0.6, 20.13±1.2, 23.21±0.8 and 26.10±1.0 mM. Finally, docking studies revealed that the synthesised compounds have similar binding modes against the prospective biological targets. This work introduces compound 8a as promising anti-cancer agent.