Problem statement: Propolis samples from tropical zones, such as Java (Indonesia) with its vast biodiversity, have become a subject of increasing scientific and economic attention. The association of the chemical composition of propolis from different geographic regions with cytotoxic activities lead to the identification of active principles, a fundamental tool to achieve standardization of this bee product. Approach: The purpose of this research was evaluate the quality of propolis collected at different places in Java (Indonesia) based on cytotoxic activity. The ethanolic extracts of propolis from different areas in Java were tested for cytotoxicity against tumor cell lines (T47D, MCF-7, Hela, Myeloma and Vero) using MTT assay. Propolis samples were collected from Batang (Central Java), Lawang (East Java) and Sukabumi (West Java). Results: The extract of propolis from Batang showed the most potent activity of T47D and MCF-7 with IC 50 34.67±8.3 and 37.8±2.5 µg mL −1 . The extract of propolis from Sukabumi showed the most potent activity of Hela cell with IC 50 147.34±8.9. However, all propolis extract did not show activity of myeloma and Vero cells. Conclusion: Ethanolics extract of three propolis samples from Batang (Central Java), Lawang (East Java) and Sukabumi (West Java) regions in Java were investigated using GC-MS. From 37 compounds identified, 7 among of them were found for the first time in propolis. This indicated that the secondary metabolite extract of propolis from Batang (Central Java) obtained in the study has antiproliferative activity of breast carcinoma cells (T47D and MCF-7).