Comblike poly(ethyleneimine) (PEI) with -(CH2CH2)N(CH2CH2NH2)-as the repeating unit has been synthesized by employing a protecting group strategy, and analyzed by potentiometric titration. If the degree of protonation is plotted as a function of pH, the titration curve shows three protonation steps separated by two intermediate plateaus, namely one at a degree of protonation of 1/2 and a second one at 3/4. The first protonation step occurring at pH around 9.5 corresponds to the protonation of the primary amine groups on the side chains. During the second protonation step at pH around 4.5 every second tertiary amine protonates. The final protonation step, where the remaining tertiary amines protonate, is suspected to occur in a pH range near 0. The latter point and the detailed protonation mechanism are deduced on the basis of a site-binding model. The model, which has been independently calibrated on titration data of low molecular weight amines, is capable to predict the titration curve quantitatively to a good degree of accuracy.