Abstract Background Moschus is a rare and precious natural medicine. Due to the properties of resources scarcity and expensive price of natural musk, artificial musk has been developed as substitute materials in some prescriptions. Rapid and accurate identification of natural or artificial musk in complex traditional Chinese medicine (TCM) preparations is also a challenge. Method A strategy from non-targeted to targeted gas chromatography-mass spectrometry (GC–MS) metabolomics was developed for discrimination of natural and artificial musk. Firstly, GC–MS-based non-targeted analysis combined with chemometrics was used to find the potential chemical markers to distinguish natural musk and artificial musk. Subsequently, targeted metabolomics was used to analyze musk in preparations with multiple reaction monitoring (MRM) mode by use gas chromatography coupled with triple quadrupole mass spectrometry (GC-QQQ MS). Results Two chemical markers named prasterone and androsterone have been selected and could be detected in all Compound Pien Tze Huang preparations (CPZHs) containing artificial musk, while the CPZHs containing natural musk did not detect two markers with S/N (signal to noise ratio) less than 3. Conclusion Our work provides an applicable approach to select the practical chemical markers for the assessment of musk in preparations to realize the traceability of musk in TCM and improve the quality control of musk-containing preparations.