It has been reported that this cytotoxic potential of the leaves cardenolide compound can inhibit the growth of breast cancer cell MCF-7, skin cancer cells KB, and lung cancer cells NCL-H18 (Seeka and Sutthivaiyakit 2010). increased expression of caspase-8. Keywords: Calotropis gigantea, calotroposid A, apoptosis, WiDr cell line, cell cycle arrest Introduction Calotropis gigantea (C.gigantea) is a herb that grows wild and is widespread in Indonesia. For generations, this herb has been traditionally used by the people of Indonesia to cure rashes, scabies, boils, cough, trachoma, constipation (the leaves), asthma, nausea, stomach pain (the flowers), syphilis, viper bites (the roots), toothache, swelling, ear inflammation, intestinal worms infestation and dysentery (Radjakmanugunsudarso 1968). Scientific evidence on anticancer activity happening in this herb has been reported to include calotropon compound of the roots that induce cytotoxic activity against leukemia cell K562 and gastric cancer cells 7901 (Wang et al., 2008). The methanol extract and chloroform fraction of its flowers induce antitumor activity on mice to ht ascites carcinoma (Habib et al., 2010). The methanol extract (ME) and chloroform fraction of the roots of C. gigantea is usually capable of inhibiting the growth of ascites carcinoma by 43.90% (20mg ME/kg) and 57.07% (40mg CF/kg) (Habib and Karim, 2011). It has been reported that this cytotoxic potential of the leaves cardenolide compound can inhibit the growth of breast cancer cell MCF-7, skin cancer cells KB, and lung cancer cells NCL-H18 (Seeka and Sutthivaiyakit 2010). The dichloromethane cytotoxic extracted from the leaves is usually potential to IL20RB antibody fight breast cancer cells MCF-7 and MDA-MB-231, Hela cells, colon cancer cells HT-29, ovarian cancer cells Skov-3 and also liver cancer cells Hep-G2 (Wong et al., 2011) . BMS 433796 In previous studies, it has also been reported that this ethanol extracted from C. gigantea leaves was capable of inhibiting the in vivo growth of fibrosarcoma at the dose of 100 and 150 mg/kg with an increased expression mechanism of caspase-3 (Muti et al. 2016). C.gigantea root extract contained higher anticancer activity than its leaves and flowers (Mutiah et al., 2016). The ethyl acetate fraction of the leaves (IC50 41.79 g/ml) and its dichloromethane fraction BMS 433796 (IC50 40.57g/ml) had higher cytotoxic activity than those of butanol fraction (IC50 737.74 g/ml) and water (IC50 8493 g/ml) (Mutiah et al., 2017). In a preliminary test around the fractionated ethanol extract of the roots of calotropis gigantea, the ethyl acetate fraction (IC50 BMS 433796 0.063 g/ml) showed higher cytotoxic activity than that of dichloromethane fraction (IC50 0.367 g /ml), butanol (IC50 12:18 g/ml) and water (IC50 8493g/ml). Based on these data, it can be seen that this ethyl acetate fraction of C. giganteas roots performed a higher anticancer activity than the other fractions. It was presumed that this ethyl acetate fraction contained in the active compound was responsible for the anticancer activity around the roots of C. gigantea. Therefore, it is important to conduct further research around the ethyl acetate fraction on C giganteas roots, which is mainly related to the active compounds comprised in these fractions and the operating mechanism of the active compound. In this study, an isolation on anticancer compound from ethyl acetate fraction of the roots of C. gigantea had been conducted which was active towards WiDr colon cancer cells. The isolation was conducted by using bioassay guided isolation approach method. The isolation was conducted based on anticancer activities, ranging from fractions, sub-fractions and isolates. Then, the chemical structures of the active isolates were decided. This research is usually important because there are still many active anticancer compounds from the roots of C. gigantea which are left undiscovered. In addition, the approach taken by previous researchers have not been based on bioassay guided isolation. This step is important to take as it determines.