Rumex crispus (RC) or Cordyceps militaris (CM) has been used traditionally to treat various diseases and has been also consumed as a functional food made by humanitas medicine concept. We prepared a new herbal mixture, AST2017-01 which is mainly composed of processed (Beopje)-RC (P-RC) and -CM (PCM). This study aims to validate marker compounds (chrysophanol and cordycepin) in P-RC and P-CM and water extracted-RC and -CM using liquid chromatography-tandem mass spectrometry. In addition, we analyzed contents of chrysophanol and cordycepin in AST2017-01. The linarites of chrysophanol and cordycepin were obtained in calibration curve with a coefficient of correlation of 0.999. The results showed that the concentrations of chrysophanol and cordycepin in P-RC and P-CM were almost 1.70 and 1.23 fold higher than that in RC and CM, respectively. Furthermore, contents of chrysophanol and cordycepin in the AST2017-01 are approximately 0.13% and 0.028%, respectively. In conclusion, these results indicate that chrysophanol and cordycepin were validated as marker compounds in the AST2017- 01.