Objective In order to establish an experimental method applicable to the determination of GI in honey-containing herbal preparations in mice. Methods with reference to WS/T 652-2019, the experimental method for mouse testing was optimized on the basis of the target amount of the tested food for mice of 0.1 g and the time interval of 72 h between the independent measurements, and the samples of different proportions of fructose glucose syrups were used to validate the feasibility of the initially selected conditions. The optimized method was applied to the determination of GI in honey and the honey-containing Chinese medicinal preparation Guhan Nutritive Essence. Results The results showed that under the optimized conditions, the GI values of fructose-glucose syrup were the closest to the theoretical values, and the correlation with the fructose ratio was highly linear (R2=0.9959). The GI values of the four honeys with different fructo-glucose ratios were 60.3, 54.3, 56.5, and 59.3, which were lower than those of fructo-glucose syrups with the same fructo-glucose ratio. The GI values of the honey-containing herbal preparations, Gu Han Nutritive Essence, ranged from 45.2 to 59.6 and decreased with the decrease of honey GI. Conclusion These results suggest that the optimized mouse assay is expected to be an effective tool for GI determination of honey-containing herbal preparations and screening of honey raw materials.