Before performing mutagenesis on the OPH bound to paraoxon, we used docking and ligand-based methods, combined with molecular dynamics (MD) simulations, to model possible binding modes of paraoxon in the OPH binding site. Overall, we found a stable binding pose of paraoxon, which differs from the native binding mode of the substrate analog. Moreover, using the predicted binding mode, we performed MD simulations on different OPH mutants and found three different residues, D253, H254, and I255, involved in the stability of the catalytic site. Here, we report a detailed computational structural analysis on five mutant complexes including these residues (D253E, D253E-H254R, D253E-I255G, H254S, and I255S).