The proton affinities of lysine (1) and its three homologues ornithine (2), 2,4-diaminobutanoic acid (3), and 2,3-diaminopropanoic acid (4) have been determined using two different variants of the extended kinetic method in an electrospray ionization-quadrupole ion trap instrument. A value of 1004.2 +/- 8.0 kJ/mol is recommended for the proton affinity for lysine on the basis of this work and previous experimental measurements and theoretical predictions. Values of 1001.1 +/- 6.6, 975.8 +/- 7.3, and 950.2 +/- 7.1 kJ/mol have been determined for the proton affinities of 2-4. These experimental results are supported by hybrid density functional theory calculations at B3LYP/6-311++G**//B3LYP/6-31+G*. An analysis of the derived entropy terms lends support to the notion that these values can be used as a quantitative prediction for the thermodynamic entropy of protonation provided that appropriate error bars are assigned. Finally, for systems in which this entropy term is large, it is essential that the extended kinetic method be used to derive accurate proton affinities.