Precise binding free energy predictions for ligands targeting metalloproteins, especially zinc-containing histone deacetylase (HDAC) enzymes, require specialized computational approaches due to the unique interactions at metal-binding sites. This study evaluated a docking algorithm optimized for zinc coordination to determine whether it could accurately differentiate between protonated and deprotonated states of hydroxamic acid ligands, a key functional group in HDAC inhibitors. By systematically analyzing both protonation states, we sought to identify which state produced docking poses and binding energy estimates most closely aligned with experimental values. The docking algorithm was applied across HDAC 2, 4, and 8, comparing protonated and deprotonated ligand correlations to experimental data. Results demonstrated that the deprotonated state consistently yielded stronger correlations with experimental data, with R² values for deprotonated ligands outperforming protonated counterparts in all HDAC targets (average R2 = 0.80 compared to the protonated form where R2 = 0.67). These findings emphasize the significance of proper ligand protonation in molecular docking studies of zinc-binding enzymes, particularly HDACs, and suggest that deprotonation enhances predictive accuracy. The study’s methodology provides a robust foundation for improved virtual screening protocols to evaluate large ligand libraries efficiently. This approach supports the streamlined discovery of high-affinity, zinc-binding HDAC inhibitors, advancing therapeutic exploration of metalloprotein targets. A comprehensive, step-by-step tutorial has been provided to facilitate a thorough understanding of the methodology and enable reproducibility of the results.