The full details on how to select correct pK a values and polynomial coefficients can be found in our previous GROMACS constant pH publications. After the structure and parameters for the constant pH MD simulation are automatically generated by phbuilder (and modified by the user if needed), the system needs to be minimized and equilibrated, after which the constant pH MD simulation can be started. In addition to the tools described before, three external scripts are provided: (1) create parameterization.py to set up a parameterization of novel single-state compounds, (2) fit parameterization.py to compute the polynomial coefficients of V MM fit from the parameterization runs, and (3) create titration.py to set up titration runs of the equilibrated systems. Both the titration and parameterization require multiple constant pH simulations of the same system with different input conditions (pH in case of titration, fixed λ-values in case of parameterization).