Juvenile hormone (JH III) is an important example of a chemical signaling derived from the terpene biosynthetic pathway. Because of its impact on many species’ reproductive and developmental processes, analytical approaches must be developed to accurately identify and measure it in biological samples. In this technical note, we explored the gas-phase dissociative behavior of JH III using tandem mass spectrometry combined with electrospray ionization to provide diagnostic fragment-ions for quantitative purposes.