¹Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Universitas Airlangga, Surabaya 60115, Indonesia DOI : 10.35333/jrp.2020.138 Breast cancer and cervical cancer were ranked first and fourth of the leading cause of death of women in the world. One of natural compounds that was reported on having cytotoxic activity against various cancer cells with high selectivity is betulinic acid. In our previous study, it was found that the acetone extract of Solanum mammosum callus culture contains betulinic acid. The aims of the current study were firstly to investigate the anticancer activity of the acetone extract of S. mammosum as well as betulinic acid, and secondly to quantitatively determine the betulinic acid content in the acetone extract of S. mammosum culture. The anticancer activity was carried out using the MTT method against HeLa and Vero cells. The determination of betulinic acid content was done using the TLC densitometry method. The results showed that the acetone extract of S. mammosum gave anticancer activity against HeLa cancer cells with IC50 value of 120.5 μg/mL, however, it was less active compared to betulinic acid with IC50 of 19.3 μg/mL. Both samples were found to be non-toxic on Vero normal cells. Based on TLC densitometry, the acetone extract of S. mammosum contain 1.2% w/w betulinic acid. Keywords : Solanum mammosum; callus culture; anticancer; HeLa; Vero, betulinic acid