Abstract: A method of rapid resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (RRLC-Q-TOF-MS) was developed for the simultaneous determination ginsenoside Rh1 enantiomers，and the pharmacokinetics of rats were studied. 20-(S) and 20-(R) ginsenoside Rh1 were separated on Agilent SB C18 column, using wateracetonitrile（90∶10，V∶V）and water-acetonitrile（10∶90，V∶V），including 15 mmol/L ammonium formate as mobile phase A and B. The flow rate was 0.3 mL/min, and the injection volume was 5 μL. Determination was carried out by Q-TOF-MS in negative ion mode. The results show that the linearity of 20-(S) and 20-(R) ginsenoside Rh1 are 0.24—39.00 mg/L and 0.035—7.80 mg/L， respectively. The limits of quantification (LOQ) are 0.20 and 0.03 mg/L， the limits of detection (S/N=3) are 0.10 and 0.02 mg/L， respectively. The intra-day precisions are 89.54%—95.79% and 88.76%—94.33%, and relative deviations are less than 5%. The inter-day precisions are 88.16%—92.41% and 88.49%—94.35%, and the relative deviations are less than 5%. The recoveries of low, medium, and high concentrations are more than 90%. The method can be applied in separation and the pharmacokinetics studies of ginsenoside Rh1 enantiomers in rats.