Abstract: Global DNA methylation in tissue was determined by liquid chromatography-tandem mass spectrometry （LCMS/MS）. DNA was extracted by phenol-chloroform, hydrolyzed using 88% formic acid at 140 ℃, spiked with cytosine-2, 4-¹³C₂,¹⁵N₂ as internal standard, reconstituted in methanol and analyzed by LC-MS/MS with multiple reaction monitoring mode, to reflect the global DNA methylation level of the tissue. Results show that the limit of quantification is 1 μg•L^-1 for both Cytosine (Cyt) and 5-methylcytosine (5mCyt), and the linear ranges of calibration curve are 1—50 μg•L^-1 and 1—100 μg•L^-1 for 5mCyt and Cyt， respectively, with correlation coefficient higher than 0.99. The relative standard deviations (RSDs) are 0.70%—4.09% and 0.60%—4.81% for Cyt and 5mCyt， respectively. The intra-day precision expressed as RSD ranges from 1.86% to 4.67%, while the inter-day values from 3.72% to 4.68% .The recovery ratio of method varies from 86.52% to 105.14%. The method is simple， and can be used for detection of Cyt and 5mCyt, thus enabling the evaluation of global DNA methylation.