Juvenile hormone (JH) III is responsible for control of a variety of insect physiological and developmental states, including caste differentiation of the Formosan subterranean termite (Coptotermes formosanus Shiraki). We report here a simplified, efficient sample preparation and an optimized LC-ESI-MS method for quantifying JH III in whole body extracts. Sample preparation comprises hexane extraction (from termite whole bodies) and C18 cartridge purification. Previous LC-ESI-MS protocols exhibited the following two problems: (1) ion fragmentation differed when comparing spectra from insect samples and authentic JH III and (2) a JH III monitoring ion was not resolved from other unknown compounds in whole body samples from termites. To overcome these problems, we used a pentafluorophenyl LC column and water/acetonitrile containing ammonium acetate as solvent. In a mass chromatogram (m/z 235) of termite samples, a peak was detected at the retention time of authentic JH III, and MS(2) of this peak confirmed that the ion is a fragment of JH III, [M-CH(3)OH+H](+), being the base peak in both termites and authentic JH III samples. The protocol enables quantification of JH III in a single termite with signal/noise >10:1 and the limit of quantification is 21pg.