Abstract: Hydrogen nanobubbles have shown great potential in various applications such as environmental remediation, energy fuel production, and medical applications. However, accurately determining the total hydrogen content in hydrogen nanobubble water has been a challenge, which affects the evaluation of nanobubble generator performance. To address this issue, a method using headspace gas chromatography was developed, which transferred hydrogen from the liquid phase to the gas phase by heating the headspace bottle to achieve gas-liquid equilibrium, and then determined the total hydrogen content in hydrogen nanobubble water using gas chromatography. The results show that the optimal equilibrium condition for sample pretreatment is 50 ℃ for 15 min. It has been confirmed that the headspace gas chromatography method is suitable for analyzing a wide range of hydrogen nanobubble water systems (with number concentrations ranging from 10⁶ to 10⁸ mL⁻¹ and average particle sizes ranging from 100 to 300 nm) prepared by most nanobubble generation methods, by detecting both number concentration and particle size distribution of hydrogen nanobubbles in hydrogen nanobubble water. When the number concentrations of hydrogen nanobubbles are within the range of 6.7×10⁶-3.8×10⁸ mL⁻¹, the corresponding measured hydrogen contents fall between 0-3.12 mg/L. The changes in number concentrations of hydrogen nanobubbles are consistent with the corresponding hydrogen contents, and the detection process can be completed within 2 minutes. This method is simple and efficient, and can fulfill the test requirements regarding different number concentrations of hydrogen nanobubble water, providing a feasible option for quantifying the total hydrogen content in hydrogen nanobubble water. It facilitates further research on their significant applications of hydrogen nanobubbles in various fields and enables the evaluation of nanobubble generator performance.