An high-performance liquid chromatography (HPLC) method for identification of quaternary and non-quaternary compounds (parent compounds, intermediates, by-products, and products) within the synthesis of the acetylcholinesterase reactivator HI-6, the most promising antidote of nerve agent poisonings, is described. This HPLC method could be of high interest as a quick purity control for those who are interested in development of new acetylcholinesterase reactivators as well as for those who are interested in the synthesis of HI-6 in laboratory or in large-scale production. An HPLC method for quaternary compounds without using common ion-pairing reagents was developed, too.