Abstract A convenient procedure is described for the isolation and quantitative determination of juvenile hormones from biological material by their conversion into the 10-heptafluorobutyryloxy-11-methoxy derivatives. After precipitation at low temperature, the lipid is separated by thin-layer chromatography (TLC), and the isolated hormone fraction is converted into the 10-hydroxy-11-methoxy derivatives which are purified by high-performance liquid chromatography (HPLC). These derivatives are treated with heptafluorobutyrylimidazole and the resulting derivatives are finally separated and quantified by electron capture gas-liquid chromatography. Overall yields are 20–50% with 1–4 g insects extracted. Marker substances used are triphenylmethanol for HPLC, and bis(ethylhexyl) phthalate and dibutyl phthalate for TLC and HPLC.