New thiazolo[4,5-d]pyrimidine analogues were synthesized and biologically assessed in-vitro for their antineoplastic activity. The growth inhibitory effects of these compounds were assessed through the National Cancer Institute-United States of America (NCI-USA) anticancer screening program. Compound5(7-Chloro-3-(2,4-dimethoxyphenyl)-5-methylthiazolo[4,5-d]pyrimidine-2(3H)-thione) was found to have a potent and broad-spectrum cytotoxic action against NCI panel with GI (50% growth inhibition concentration) mean graph midpoint (MG-MID) = 2.88 µM. MTT assay was used to determine IC values of the most potent agent against HCT-116 colorectal carcinoma and WI-38 human lung fibroblast cell lines; 5.33 µM ± 0.69 and 21.69 µM ± 1.04, respectively. Flow cytometric analysis revealed that compound5triggered apoptosis and G2/M cell cycle arrest. The ability of compound5to inhibit CDK1 (Cyclin-Dependent Kinase 1)/Cyclin B complex was evaluated, and its IC value was 97 nM ± 2.33. Moreover, according to the gene expression analysis, compound5up-regulated p53, BAX, cytochrome c, caspases-3,-8 and-9 besides down-regulated Bcl-2. In conclusion, compound5exerted a potent pro-apoptotic activity through the activation of the intrinsic apoptotic pathway and arrested the cell cycle at the G2/M phase.