ProtoCaller is a Python library distributed through Anaconda which automates relative protein-ligand binding free energy calculations in GROMACS. It links a number of popular specialized tools used to perform protein setup and parametrization, such as PDB2PQR, Modeller, and AmberTools. ProtoCaller supports commonly used AMBER force fields with additional cofactor parameters, and AM1-BCC is used to derive ligand charges. ProtoCaller also comes with an extensive PDB parser, an enhanced maximum common substructure algorithm providing improved ligand-ligand mapping, and a light GROMACS wrapper for running multiple molecular dynamics simulations. ProtoCaller is highly relevant to most researchers in the field of biomolecular simulation, allowing a customizable balance between automation and user intervention.