<div> <div> <div> <p>Alchemical free energy calculations are an increasingly important modern simulation technique. Contemporary molecular simulation software such as AMBER, CHARMM, GROMACS and SOMD include support for the method. Implementation details vary among those codes but users expect reliability and reproducibility, i.e. for a given molec- ular model and set of forcefield parameters, comparable free energy should be obtained within statistical bounds regardless of the code used. Relative alchemical free energy (RAFE) simulation is increasingly used to support molecule discovery projects, yet the reproducibility of the methodology has been less well tested than its absolute counter- part. Here we present RAFE calculations of hydration free energies for a set of small organic molecules and demonstrate that free energies can be reproduced to within about 0.2 kcal/mol with aforementioned codes. Achieving this level of reproducibility requires considerable attention to detail and package–specific simulation protocols, and no uni- versally applicable protocol emerges. The benchmarks and protocols reported here should be useful for the community to validate new and future versions of software for free energy calculations.</p></div></div></div>