Objective To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of ginsenoside Rh2 (GRh2) in plasma of mice, so as to provide preclinical data support for the pharmacokinetic study and application of GRh2. Methods C57BL16 mice were given 7.5 mg/kg GRh2 by gavage. After administration, 0.03 mL of whole blood was collected at 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h. Then, the whole blood was centrifuged and the serum was treated with 0.1% formic acid acetonitrile, dried by nitrogen (40 ℃), and redissolved with 50.0 μL 50% methanol solution containing 100 ng/mL diclofenac sodium. After vortex mixing for 5 min at room temperature, it was put into the automatic sampler for sampling analysis. The chromatographic column was Waters BEHC₁₈ (2.1 mm×50.0 mm, 1.7 μm), the mobile phase was aqueous solution containing 0.1% formic acid and acetonitrile solution containing 0.1% formic acid at a flow rate of 0.60 mL/min, the column temperature was 40 ℃, and the injection volume was 1.00 μL. The electric spray ion source, positive ion mode and multiple reaction monitoring mode were performed. A standard curve was established to calculate blood drug concentration. The blood drug concentration-time curve was established according to the blood drug concentration, and the main pharmacokinetic parameters were calculated. Results The linear range of the standard curve of drug containing plasma was 100-40 000 ng/mL, and the correlation coefficient (r) was 0.996 0. After internal standard normalization, the matrix effect factors of GRh2 were 1.09, 1.06, and 1.00 (between 0.8 and 1.2), indicating no significant matrix effect. The precision and accuracy results showed that the average measured concentration of GRh2 samples at each concentration level was 103, 333, 23 800 and 35 000 ng/mL, the inter batch standard deviation was 6.47-1 120 ng/mL, the inter batch relative standard deviation was 1.5%-8.3%, and the inter batch accuracy deviation was 93.3%-111.1%. The long-term stability, short-term stability, repeated freeze-thaw property, and extraction recovery rate of GRh2 were all good. The pharmacokinetic parameters T_max and C_max of GRh2 in mice were (1.42±1.01) h and (1 251±495) ng/mL, respectively, indicating that the absorption and utilization rate of GRh2 in vivo was high and GRh2 had good drug performance. Conclusion The established LC-MS/MS method is accurate and reliable, and can be used to determine the concentration of GRh2 in mouse plasma and study its pharmacokinetic.