High performance thin layer chromatography hyphenated with mass spectrometry (HPTLC-MS) was implemented for the first time for the simultaneous tracking and quantitation of the major flavonolignans in fruit extracts of milk thistle (Silybum marianum L.) varieties; marianum and albiflora.  A mobile phase of chloroform: acetone: formic acid (110: 16.5: 8.5 v/v/v) was utilized for the development of the tracked flavonolignans followed by their visualization under UV 366 nm after NP/PEG derivatization. The tracked flavonolignans were identified, using mass analysis of the developed flavonolignans directly from the developed plate, and quantified. The developed method's linearity, accuracy, and precision were all confirmed in accordance with ICH requirements. Linear calibrations for the quantified flavonolignans were obtained with a correlation coefficient > 0.98. Precision was confirmed with a relative standard deviation ranging between 1.34 and 1.83%.  In addition, acceptable accuracy was obtained with recovery exceeding 97%, as well as appropriate limit of detection (LOD) and limit of quantitation (LOQ).