Juvenile hormone (JH) is an important insect hormone known to have many effects on development, reproduction, and behavior in both solitary and social insects. A number of questions using ants as a model involve JH. This procedure allows for quantification of circulating levels of JH III, which can be an important factor in many questions relating to insect research. The JH III is extracted from a subject, purified, and converted to a d(3)-methoxyhydrin derivative that can be quantified by gas chromatography-mass spectrometry (GC-MS). The major advantages of this protocol are its high resolution, and its ability to quantify significant differences between relatively small quantities of the hormone. Its major limitations are the time necessary to process samples, its relatively high cost, and maintaining the sensitivity of the equipment.