Myco-Off Mycoplasma Cleaner is a kit for removing Mycoplasma in cell culture, serum, and medium. Unlike conventional antibiotics, this kit kills mycoplasma by disrupting the membrane structure, and can remove antibiotic-resistant mycoplasmas, so as to achieve more effective elimination of mycoplasma rather than inhibiting proliferation. This kit is also effective on Gram-negative and postitive bacteria. Our results showed that all mycoplasmas, located both inside and outside cells, were successfully removed after a incubation with MycoOff Mycoplasma Cleaner for 3-7 days. No further re-contamination was found for 4 months after one treatment with this kit. This kit can be used to kill most kinds of mycoplasmas and is not toxic to cells. This kit has been validated in most commonly used cell lines, including mouse amd human embryonic stem cells or iPS cells, HEK293, Hela, HepG2, HCT116, COS-7, Vero, Huh-7, MDCK, PANC-1, SW620, and U2OS.