Introduction: Tumor diseases are a leading cause of death worldwide and there is a need to discover an efficient form of tumor treatment. According to recently shown pharmacological activity, purine nucleoside analogs (PNA) deserve attention and further research. Study aim: To determine the cytotoxic effect of N-9 sulfonyl purine on tumor and normal cell lines and to determine a possible mechanism behind cell deaths. Materials and methods: The cytotoxic effect of 14 newly synthesized N-9 sufonyl purines were identified on a panel of human tumor cells (HeLa, CaCo-2, NCI-H358, K562, Raji, HUT-78, CCRF-CEM, MOLT-4), normal human fibroblasts (BJ) and normal epithelial cells of a dog's kidney (MDCK). After the treatment, which involved the previously mentioned compounds, cell viability was determined by a MTT test and the cytotoxic effect was confirmed by measuring the LDH enzyme in the cell culture medium. The mechanism behind cell death was examined by measuring the caspase-3 enzymatic activity in the cell lysate, while the apoptotic pathway was determined by measuring the mitochondrial membrane potential by means of flow cytofluorometry. Results: The examined N-P sulfonyl purine shows the cytotoxic effect depending on the applied concentration and cell line. Based on the results, cytotoxicity was expressed on human leukemia and lymphoma cell lines and compounds R-191 and D7-23 were isolated. The previously mentioned compounds did not show caspase-3 activity, but the mitochondrial membrane potential was distorted. Conclusion: Based on the obtained results we can conclude that certain newly synthesized N-9 sulfonyl purines show high anti-proliferative and cytotoxic activity, especially on the human leukemia and lymphoma cell lines. The absence of caspase-3 activity demands further detailed research and confirmation of a caspase-independent apoptotic pathway.