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Objective To develop a highly effective HPLC method for standardization of silymarin and for characterization of minor components,which has not been reported previously.Methods An HPLC fingerprinting method and 1H-NMR spectroscopy were employed to provide assurance for a standardized silymarin product (MK-001).Results A total of 18 marker compounds were identified and characterized including ten flavonolignans and eight small molecules including adenine,adenosine,uridine,and 3,5,7-trihydroxychromone which were first isolated and characterized from silymarin.The HPLC fingerprinting method and 1H-NMR had been established for complete assignments of HPLC peaks with intensity over 1.0％.Conclusion The successfully established HPLC fingerprinting method and 1H-NMR could be applied for the standardization of commercial silymarin products.MK-001represents the first standardized silymarin with the highest content of flavonolignans ( ＞ 90％).