Heterotopic tri-cationic receptors based on 4,10,16-triaza-18-crown-6 are capable of efficient and selective binding of the zwitterionic form of 5-aminovaleric acid (5-AVA) in aqueous/methanol solution. The cooperative participation of both cation and anion binding domains of this receptor in 5-AVA complexation was established by 1H NMR experiments and DFT calculations. Heterotopic receptors and fluorescein were used for the preparation of indicator displacement assay and this assembly was applied to selective optical sensing of 5-aminovaleric acid.