To evaluate the influence of the employed partial charge distribution on the observed scores within ESP-Sim, ESP similarities were computed for the same molecule at the same geometry but with different partial charges. As ground truth, quantum-mechanically (QM) obtained RESP charges at a high level of theory (MP2 with a polarizable PVTZ basis set) were used [30], on a selection of about 3000 neutral molecules. RESP charges are specifically designed to reproduce the electrostatic potential of a molecule, so that a comparison of electrostatic potentials obtained from different charge distributions to the QM RESP charges allows for a detailed assessment of the quality of each approach for similarity comparisons. We evaluated Gasteiger (default in RDKit), MMFF94, and AM1-BCC partial charges, as well as a machine learning model (ML). The ML partial charge model is provided with the ESP-Sim package on Github. Table provides an overview of the observed mean absolute deviations of the respective partial charges from the RESP charges, as well as the ESP similarities evaluated via Carbo or Tanimoto similarities. We find that AM1-BCC charges reproduce the QM electrostatic potentials best, followed by the deep learning model, MMFF, and Gasteiger. Figure depicts a heatmap of the QM atomic charges compared to Gasteiger, MMFF, ML and AM1-BCC charges. Although there is no perfect correspondence of QM charges to any of the evaluated charges, we find highest agreements for AM1-BCC charges. Notably, Gasteiger and ML charges lead to narrower ranges than MMFF and AM1-BCC charges. This is also reflected in the Carbo and Tanimoto ESP similarities in Table . The Carbo metric, which is largely insensitive toward the magnitude of a function, yields more favorable scores for ML and AM1-BCC, in contrast to the Tanimoto metric, which is more sensitive to the absolute magnitudes, thus ranking MMFF better than ML. These differences are small, though, and a comparison of observed similarities via different charge distribution in reference to QM RESP shows similar trends between all options, Figure . We can therefore assume that even Gasteiger charges lead to a fair depiction of the electrostatic potential for most molecules. In fact, benchmarking of ESP-Sim on protein-docking databases shows little dependence of ranking metrics on the employed partial charge distribution, as detailed in the following.