We have presented and validated a new implementation of λ-dynamics based constant pH molecular dynamics in the GROMACS software. Our implementation combines several developments in this field into a single MD program, including the multisite representation of titratable groups, 38 charge interpolation, Particle Mesh Ewald electrostatics, and charge constraints. Test calculations on amino acids and proteins suggest that the new implementation is efficient, accurate and agnostic to force fields. Combined with user-friendly parameterization protocols, presented in the accompanying paper, we expect that this im- one of the protonation states of a residue, while α k i = 0 for all other λ i -coordinates. If σ k (λ) is a constraint for keeping the overall charge constant, α k i = N i atoms j q B j,i -q A j,i , with N i atoms the number of atoms whose charges change as a function of λ i .