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 1 H-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 1 H-NMR had been established for complete assignments of HPLC peaks with intensity over 1.0%. Conclusion The successfully established HPLC fingerprinting method and 1 H-NMR could be applied for the standardization of commercial silymarin products. MK-001 represents the first standardized silymarin with the highest content of flavonolignans ( > 90%). 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 1 H-NMR spectroscopy were employed to provide assurance for a standardized silymarin product (MK- 001). Results A total of 18 marker compounds were identified and 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 1 H-NMR had been established for complete assignments of HPLC peaks with intensity over 1.0%. Conclusion The successfully established HPLC fingerprinting method and 1 H-NMR could be applied for the standardization of commercial silymarin products. MK-001 represents the first standardized silymarin with the highest content of flavonolignans (> 90%).