A mass spectrometric method is presented that facilitates the identification and differentiation of primary, secondary and tertiary amino functionalities in protonated monofunctional analytes. This method utilizes gas-phase ion–molecule reactions of protonated analytes with neutral hexamethylphosphoramide (HMPA) and diethylmethylphosphonate (DEMP) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR). A variety of protonated analytes containing different functional groups, namely, amino, amido, N-oxide and various oxygen-containing functional groups, were examined to demonstrate that protonated primary and secondary amines can be identified and differentiated by reactions with HMPA and DEMP. However, differentiation of tertiary amines from some N-oxides requires additional experiments. First, protonated secondary and tertiary amines can be differentiated from protonated primary amines, amides and oxygen-containing functionalities, as well as from each other (but not from protonated N-oxides), by using HMPA. Protonated primary amines, amides, some N-oxides and oxygen-containing analytes, most with a proton affinity (PA) < 229 kcal/mol, only transfer a proton to HMPA (PA = 229 kcal/mol). In contrast, protonated secondary amines also form two stable hydrogen-bound adducts (MH + + HMPA, MH + + 2HMPA; M: amine), and tertiary amines and some N-oxides (with PA ≥ 222 kcal/mol) react with HMPA by forming just one stable hydrogen-bound adduct (MH + + HMPA). Further, ion–molecule reactions with the other reagent, DEMP, allow the differentiation of protonated primary and secondary amines from tertiary amines and N-oxides and from protonated oxygen-containing analytes and amides. Protonated primary amines and secondary amines (most with PA ≥ 220 kcal/mol) react with DEMP (PA = 219 kcal/mol) by forming two stable hydrogen-bound adducts (MH + + DEMP and MH + + 2DEMP), while protonated oxygen-containing analytes and amides (with PA ≤ 221 kcal/mol) solely transfer a proton to DEMP. Protonated tertiary amines and N-oxides react yet differently, and yield only one stable hydrogen-bound adduct (MH + + DEMP) with DEMP. Protonated N-oxides can be differentiated from protonated tertiary amines by using previously reported methods based on diagnostic ion–molecule reactions of protonated N-oxides with 2-methoxypropene, dimethyl disulfide and/or tri(dimethylamine)borane.