A method was developed to quantify diaminomaleonitrile (DAMN) in complex aqueous matrices using high performance liquid chromatography with UV and electrospray ionization mass spectrometric (ESI-MS) detection. The method is rapid (analytical times <10 min), able to quantify DAMN against complex backgrounds, and achieved with part per billion detection limits. Given the central role of DAMN in generating heterocycles of known biological significance, this study provides a rapid and sensitive method for quantifying DAMN in complex solutions.