Synthesis of novel quinazolinone derivatives was performed from the reaction of N-benzoyl substituted piperazine-1-carbothioamide with 2-chloromethyl quinazolinone derivatives and screened for their in vitro cytotoxic activity by MTT assay. The cell lines used were NCI (human lung cancer cell), MCF 7 (Breast cancer cell), and HEK 293 (Normal epidermal kidney cell). Result of screening on cell line showed moderate to good anticancer activity for all the compounds. Compound 3d (IC50 = 1.1 ± 0.03 lM) was found to be the most active compared to standard methotrexate (IC50 = 2.20 ± 0.18 lM) and 5-florouracil (IC50 = 2.30 ± 0.49 lM). Structure activity relationship of synthesized analogs suggested that the presence of NH linker with aryl moiety at the third position of quinazolinone ring was important for potent anticancer activity. Electron donating group on phenyl ring at the third position of quinazolinone ring gave better anticancer activity then unsubstituted phenyl and electron withdrawing group. Activity by substituted piperazine at 2nd position of thiazole linked with quinazolinone scaffold gave better activity in the order of H>CH3>CO–C6H5. Our findings may impart new direction to medicinal chemists and biochemists for further investigations of quinazolinone-thiazole containing anticancer agents.