The analytical method of β-ecdysone, the insect moulting hormone, by high performance liquid chromatograph (HPLC) with UV detector was developed using boronic ester derivatization and applied to the extracts of Ajuga iva, Silene otites and Schistocerca egg. Derivatization of yield with methyl-, butyl-, and phenyl-boronate was completed under mild conditions with 20-hydroxyecdysone. The conversion ratios of boronate were estimated to be 70% in methylboronic acid, 89% in butylboronic acid and 93% in phenylboronic acid. Phenylboronate showed a high sensitivity and demonstrated an effective separation on HPLC. The optimum temperature and reaction time for derivative formation were 25℃ and 20 min. respectively. β-Ecdysone was effectively identified in extracts of Ajuga iva, Silene otites and Schistocerca egg by the HPLC method.