Mobley and Guthrie reported hundreds of molecules with experimental hydration free energy data. Notably, for these molecules, the energy estimated using the MMFF method with the traditional TI protocol demonstrated a deviation of ±1.5 kcal/mol. We randomly selected 20 compounds containing the elements C, H, O, N, F, and Cl from this dataset, applied our newly-proposed protocol to predict hydration free energies using ANI-2x in combination with GAFF2. We used ABCG2 charge model 61 to calculate the electrostatic interactions, considering it outperforms AM1-BCC in many molecular property calculations. Figure illustrates the prediction accuracy of different models. Note that the results for CGenFF 1 and GAFF 2 were directly obtained from previous publications. In summary, the overall data distributions of ANI-2x and GAFF2 are relatively similar, with nearly the same mean absolute error (MAE) of 0.47 kcal/mol and 0.41 kcal/mol (Figure ), respectively, which are significantly lower than those obtained using either CGenFF (0.84 kcal/mol) or GAFF (0.57 kcal/mol). We suspect that the slightly lower accuracy of ANI-2x compared to GAFF2 is due to some cooperation or consistency issue between them. After all, ANI-2x was trained to reproduce high-accuracy DFT energetics and forces (ωB97x-D/6-31G(d)), whereas GAFF 2 and TIP3P water were developed to reproduce both quantum mechanics and experimental data. This difference might account for the observed discrepancy. However, the quartile line distribution and mean squared error (MSE) indicate that the hydration free energies estimated by ANI-2x are closer to the experimental data. All these results indicate that our postulated theory regarding ML/MM demonstrates its comparability to the traditional TI approach in a novel way. Traditional TI, however, employs a gradual scaling-down method to reduce intramolecular interactions, which may also affect interactions between water and the molecule. This creates a highly coupled system; while our approach aims to reasonably decouple these interactions, further efforts are needed to estimate the coupling effects in TI calculations, thereby enhancing the accuracy of ML/MM TI calculations.