OBJECTIVE： To develop a detection LC-MS/MS method for global DNA methylation in medicinal plants. METHODS： Genomic DNA was isolated using plant DNA extraction kit， and then hydrolyzed by 88％ formic acid at 140 ℃. After dried with nitrogen， extracted DNA was dissolved again with mobile phase. LC separation was performed on HILIC column with mobile phase consisted of 7 mmol /L ammonium formate-acetonitrile （gradient elution） at flow rate of 0.3 ml/min. The analysis was conducted by tandem MS with positive ion electrospray ionization in multiple reaction monitoring （MRM） mode. The ratio of genomic DNA methylation in 10 commonly used medicinal plants was calculated. RESULTS： The linear ranges of Cyt and 5mC were 1-500 ng/ml （r＝0.999 5） and 0.2-100 ng/ml （r＝0.999 6）. The relative standard deviations （RSDs） of accuracy were 1.12％ and 3.68％ （n＝6）. The RSDs of intra-day precision were 2.36％ and 4.02％ for Cyt and 5mC， respectively （n＝5）. The RSDs of inter-day precision were 1.04％ and 3.54％ for Cyt and 5mC， respectively （n＝3）. The RSDs of repeatability test were 1.53％ and 3.27％ for Cyt and 5mC， respectively （n＝6）. The recoveries of Cyt and 5mC were 98.7％-102.1％ and 91.2％-103.5％. The percentages of global DNA methylation in 10 medicinal plants were ranged from 17.63％ to 25.18％. CONCLUSIONS： LC-MS/MS method is simple， rapid， sensitive and precise， and can be used for the detection of global DNA methylation in medicinal plants.