Human cytochrome P450 (CYP) 3A isoforms (mainly including CYP3A4 and CYP3A5) metabolize approximately half of the marketed drugs and thus play a critical role in the drug metabolism, due to their high abundance in the liver and broad substrate specificity.As previously reported, bufodienolides are prone to be the isoform specific probe substrates of CYP3A4, such as bufalin and resibufogenin.Bufotalin (BFT), one of the naturally occurring bufodienolides with a steroid skeleton and a unique six-membered lactone ring located at the C-17 site and an acetate located at the C-16 site, has potential pharmacological and toxicological effects such as anti-tumor activity and cardiotoxicity.