Ecdysteroids were first discovered in silkworm larvae in 1954 and are known to act as molting hormones in insects. Recently, biologically active phytoecdysone has gained increasing interest as it affects many physiological functions in mammals and has been reported to have a wide range of pharmacological properties such as protein synthesis and anticancer. 20-Hydroxyecdysone is a class of ecdysteroids commonly found in plants and animals and is known to stimulate antibody formation in humans, reduce cholesterol levels, and exert anabolic and blood sugar-lowering effects. In order to determine the content of 20-Hydroxyecdysone in Achyranthes japonica (roots) and Protaetia brevitarsis (larvae), which are known to have related benefits, we wanted to confirm their value as medicinal ingredients. The results of the analysis showed 0.0389 ug/g for Protaetia brevitarsis and 4.36 ug/g for Achyranthes japonica. These results confirm that plants secrete high concentrations of ecdysteroids to prevent insect damage, and are expected to be used as a basis for future research on the extraction of 20-Hydroxyecdysone for biological control and pharmacological use.