Bufadienolide is a kind of natural steroids with significant anti-cancer activities.Hitherto，the number of structural modification on the bufadienolides has reached more than 100.The purification of sulfated，a trace cytotoxic component bufadienolides from toad skin is a tough work due to its low content and unstable structures.However，there is no report on synthesis of bufadienolides sulfonation though they have promising cytotoxic activities.In this paper，the arenobufagin was firstly taken as an example，to develop an effective method for the synthesis of arenobufagin 3.O sulfonate.In order to enrich the arenobufagin 3.O sulfonate quickly and mildly，a reversed phase/hydrophilic solid phase extraction method based on our homemade hydrophilic Click TE-Cys was developed.The structures of arenobufagin 3.O sulfonate and arenobufagin were both identified by MS，1H NMR and 13C NMR spectra.In addition，the total bufogenins were sulfonated by this method and the sulfated bufogenins were also successfully enriched via the reversed phase/hydrophilic solid-phase extraction approach.Finally，a LC-MS system was employed to test the result,and about 20 sulfated bufogenins were primarily identified according to their molecular weight.Based on this，a systemic method was first developed for the synthesis and enrichment of the sulfated total bufogenins and a large number of potential anticancer new compounds were obtained，which provided lots of agents for further screening to achieve high effective and low toxic compounds.The efficient reversed phase/hydrophilic solid-phase extraction method for the enrichment of sulfated bufogenins could be used to enrich sulfated bufogenins in toad skin extract.