Abstract: High resolution isotope ratio mass spectrometry (HR-IRMS) is often used to measure the abundance of deuterium to verify the efficiency of isotope isolation of a cryogenic distillation column system. In order to achieve precise quantification of hydrogen isotope abundance by HR-IRMS, a fine tuning procedure was performed by using molecular hydrogen to optimize the source parameters, steering parameters and analyzer parameters. The mass resolution can reach 10 000 after tuning, which is high enough to separate all potential hydrogen and helium isotopes except for ³He⁺ and T⁺. The test results of peak center stability, peak top flatness, resolution and sensitivity stability showed that the performance of the mass spectrometer can meet the demand for hydrogen isotope analysis. The inlet pressure should be in the range of 4-33 Pa to obtain stable sensitivity, and the accelerating voltage of the ion source should not be less than 8 V because it is negatively correlated with the production rate of trimmers (H₃⁺, D₃⁺, H₂D⁺ and HD₂⁺ etc.). As the accelerating voltage decreasing, the production rate of trimers increases. Another key factor affecting the production rate of trimers is the inlet pressure, the square of which is proportional to the production rate of trimers. Therefore, optimizing the ion source parameters to obtain higher sensitivity is a better way than increasing the sample inlet pressure. On this basis, the equilibrium hydrogen isotope standards which are essential to obtain the sensitivity of each component of hydrogen isotopes were prepared by a self-made standard gas preparation device, and the sensitivity values of each component were corrected by pure gases and the self-prepared standard with verified compositions. The molecular leak rate for the inlet hole was determined by monitoring the pressure for H₂ and D₂ as a function of time as the gas leaked out, and the leak rate of the other components could be calculated using the leak rate of H₂ or D₂ because the leak rate is inversely proportional to the square root of the molar mass. Standard mixtures were analyzed to test precision and trueness of this method. The results showed that the measurement precision is better than 1% for samples with natural deuterium abundance and better than 0.5% for samples with medium to high abundance of deuterium. The measured values are in good agreement with the standard values. Compared to the low resolution mass spectrometry, this method is simple and reliable, does not need to resolve the mass overlap peak, and the accuracy of the measurement does not depend on the degree of hydrogen isotope balance in the sample.