A novel technique of insect ecdysone analysis was established by capillary zone electrophoresis using 20-hydroxy ecdysone (purity ≥93%) as a standard sample. It showed that 20-hydroxy ecdysone and other trace impurities were completely separated within 5 min with an electrolyte containing 10 mmol/L borate, at pH 9.18, 20 kV applied voltage and 0.5 psi × 5 s injected volume. Under this optimal condition, effects of azadirachtin on ecdysone in the hemolymph of Ostrinia furnacalis (Guenee) were investigated. The results showed that the peak values of 20-hydroxy ecdysone were strongly suppressed by azadirachtin and the titers remained at a lower level than those in the control samples. When compared with high-performance liquid chromatography, the primary advantages of capillary zone electrophoresis include a much shorter analysis time and detection limit and the elimination of complex pretreatment of samples.