Abstract: To identify the active components in Bufo melanostictus Schneider and clarify the difference between fresh and dried Venenum Bufonis, a UPLC-Orbitrap MS method has been established. The separation was performed with gradient elution of acetonitrile and water (with 0.1% formic acid) as mobile phase. By comparing their retention time and high resolution mass data of Venenum Bufonis extracts, 39 effective components were primarily identified by MS/MS analysis in positive ion mode. Twenty-six of them were bufadienolides. There were significant differences in the main composition between fresh and dried Venenum Bufonis. There are fewer bufadienolides in fresh toad venom.