Toad venom (named Chansu in China), derived from the skin secretions of Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider, have been widely used as Traditional Chinese Medicine (TCM) for the treatment of heart failure, sores, and pains in clinic for thousands of years.Recently, it has been found that Chansu was also increasingly applied to treat various forms of cancer, and the major active constituents are bufadienolides, a class of C-24 steroids with a characteristic-pyrone ring at C-17.The major bufadienolides from Chansu include bufalin, einobufagin, and resibufogenin, each showing significant cytotoxic activities against human liver and gastric cancer cells.In the present study, by combining the chemometric methods such as similarity evaluation and principal component analysis (PCA), we used qualitative LC/MS analysis, quantitative HPLC determination together with cytotoxic evaluation of main bufadienolides to develop a specific, practical and valid functional fmgerprint analysis approach for the comprehensive quality assessment of Chansu.