Aziridines are commonly used as reagents for the synthesis of drug substances although they are potentially mutagenic and genotoxic. Therefore, their unambiguous detection is critically important. Unfortunately, tandem mass spectrometry (MS2) based on collision-activated dissociation (CAD), a powerful method used for the identification of many unknown compounds in complex mixtures, does not provide diagnostic fragmentation patterns for ionized aziridines. Therefore, a different mass spectrometry approach based on MS3 experiments is presented here for the identification of the aziridine functionalities. This approach is based on selective gas-phase ion-molecule reactions of protonated analytes with tris(dimethylamino)borane (TDMAB) followed by diagnostic CAD reactions in a modified linear quadrupole ion trap (LQIT) mass spectrometer. TDMAB reacts with protonated aziridines by forming adduct ions that have lost a dimethylamine (DMA) molecule ([M + H + TDMAB - HN(CH3)2]+). CAD on these product ions generated diagnostic fragment ions with m/z-values 25- and 43-units lower than those of the ion-molecule reaction product ions. None of the ion-molecule reaction product ions formed from other, structurally related, protonated analytes produced related fragment ions. Quantum chemical calculations were employed to explore the mechanisms of the observed reactions.