Cholera toxin (CT) secreted by V. cholerae is the causative agent of cholera and is a major agent involved in severe diarrhoeal diseases. With the aim to create materials and production test kits for detection CT and producing antidote anti CT. Therefore, in this study, after cholera toxin was separated and purified from culture medium of V. cholerae, CT was assessed biological activity on two cells line Hepa-1c1c7 (Hepa) and H4-II-e-c3 (H4). Culture and proliferation two cell lines has been successfully gained. The test for active purifiy CT separated from the culture medium of V. cholerae on cell lines Hepa and H4 was performed. 24 hours after the cells was exposed with the CT, the cells change shape, was destroyed and die. The results reflected biological activity of CT and test for the possibility of cell proliferation after exposure with cholera toxin by MTT technique was also performed.