The juvenile hormone (JH) is a key hormone of insects. In this study, the first structure determination of JH of Rhodnius prolixus (Heteroptera; Reduviidae) known historically as the insect for which the juvenile hormone was discovered, is reported. This species is also known as a medically important pest associated with Chagas disease. The relative and absolute stereochemistry was unambiguously determined to be methyl (2R,3S)-3-((E)-6-((R)-3,3-dimethyloxiran-2-yl)-4-methylhex-3-en-1-yl)-3-methyloxirane-2-carbo xylate (JHSB(3)) by highly sensitive UPLC MS/MS method.