A new series of previously reported cytotoxic 1,3,4-thiadiazole analogs as potential anticancer agents were evaluated for induction of apoptosis. Namely, activation of caspases 3, 8 and 9 were assessed in Human Prostate Cancer Cell Line (PC-3) as well as Human Colorectal Adenocarcinoma Cell Line (HT-29). Cytotoxicity of intended compounds was evaluated in PC-3, HT-29 and Human Breast Adenocarcinoma Cell Line (MCF-7) using 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide (MTT) assay. Furthermore, activity of caspases 3, 8 and 9 were assessed in PC-3 and HT-29 cell lines and obtained results were compared to doxorubicin. Mitochondrial Membrane Potential (MMP) as well as capability of intracellular Reactive Oxygen Species (ROS) generation was also measured in these cell lines. A new series of recently reported cytotoxic 1,3,4-thiadiazole derivatives were studied mechanistically. Fortunately, the most of the investigated derivatives showed a significant increase in activity of caspases 3, 8 and 9, in PC-3 and HT-29 cell lines superior than doxorubicin. Only compound A (2-Cl) caused an increase in MMP in HT-29 cell line further than doxorubicin. None of the tested derivative did not tempted generation of reactive oxygen species in examined cell lines. Overall, the investigated derivatives could be introduced as potential anticancer lead compounds.