The gas-phase proton affinities (PA) for a series of proline-containing dipeptides have been measured in an ESI triple quadrupole instrument using the extended kinetic method (EKM).Proton affinities of ProCys (1), ProMet (2), ProSer (3), ProThr (4), ProTyr (5), ProPhe (6), and ProTrp (7) were determined to be 966.9 ± 5.3, 987.9 ± 10.4, 978.4 ± 6.1, 978.3 ± 6.1, 993.9 ± 11.2, 985.1 ± 10.7, and 998.7 ± 7.6 kJ/mol, resp.Predictions for the proton affinities for 1-7 were also obtained through isodesmic calculations at the B3LYP/6-311++G(d,p)//B3LYP/6-31+G(d) level of theory.As with a previous study on the proton affinities of ProXxx dipeptides with aliphatic side chains (Cardwell et al., Int. J. Mass Spectrom.2025, 507, 117352) the agreement between theory and experiment is mixed.For the two dipeptides with sulfur in the side chain we find excellent agreement between the EKM and calculated proton affinities.For the three dipeptides with hydroxyl groups in the side chain, the exptl. values are between 7.2 and 13.2 kJ/mol larger than the computed proton affinities.Addnl. single point energy calculations on 3-5 with B3LYP/6-311++G(2df,2p) basis sets give predicted proton affinities that are somewhat smaller than those predicted with the (B3LYP/6-311++G9d,p) basis set.MP2/6-311z++G(2df,2p) single point energy calculations on 3 and 4 give calculated proton affinities within 1 kJ/mol of the B3LYP/6-311++G(d,p) values.It appears that the EKM values for 3-5 are systematically too high, possible o to the extrapolation required to obtain the isothermal point in the EKM workup due to the large protonation entropies of the dipeptides.This result is also seen to a lesser extent for ProPhe and ProTrp in which the EKM proton affinities are 5-6 kJ/mol larger than the calculated values.As with the aliphatic ProXxx dipeptides from our previous study, we recommend using the calculated proton affinities for 1-7 with the exptl. proton affinities as upper limits.