Studies of in vitro cytotoxic activity of Sungkai leaf (Peronema canescens Jack) using a human colon and cervical cancer cells have not been reported to date; the report was still limited to bioactivity in seawater shrimp larvae. The leaves of this plant contain many secondary metabolites with potential cytotoxic activity. Plants were a source of natural medicinal ingredients for cancer treatment are known as herbal medicines. Anticancer activity of a natural substance, in vitro cell line assays, can be used. This study aimed to determine the cytotoxicity (IC50) of P. canescens leaf of the chloroform, ethyl acetate, and ethanol extract against HT-29 colon cancer and HeLa cervical cancer cells. The P. canescens leaf was extracted by the maceration method using methanol solvent. The dried sample was separated by the liquid-liquid method and successive polarity gradient eluents: chloroform, ethyl acetate, and ethanol. Cell line to used HT-29 and HeLa cells, Cytotoxicity test with MTT (3-(4,5-dimethyl azole-2-yl)-2,5-diphenyltetrazolium bromide) method. The concentration used was from 1.5µg/mL to 200.0 µg/mL. The cytotoxic activity values (IC50) of each extract on HT-29 and HeLa cells were chloroform (10.353 µg/mL and 38.913µg/mL), ethyl acetate (48.635µg/mL and 28.186µg/mL), and ethanol (42.017µg/mL), and 253.190µg/mL), respectively. The results showed that the cytotoxic activity of chloroform extracts had a strong cytotoxic effect and a moderate effect on HeLa cervical cancer cells. Anticancer research opportunities are wide open for further studies.