[Objectives]  To develop a highly efficient, stable, and accurate, method for the determination of two main ecdysteroids in insect hemolymph, namely ecdysone and 20-hydroxyecdysone (20E). Ecdysone is the immediate precursor of 20E, while 20E is the activated form of insect ecdysteroids, or molting hormones. [Methods]  Ecdysone and 20E collected in Bombyx hemolymph were separated by reverse-phase, high-performance, liquid chromatography (RHPLC), followed by an enzyme linked immunosorbent assay (ELISA), to determine their respective titers. [Results]  The ecdysone and 20E titer in Bombyx hemolymph was determined, and the relative ratio of ecdysone to 20E obtained. The biosynthesis and secretion rates of ecdysone, as well as the conversion rate of ecdysone to 20E, were calculated. [Conclusion]  The new method is highly efficient, stable, and accurate, and can be conveniently applied to determine the titers of ecdysteroids in insects.