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The genus Ajuga is used for the treatment of joint pain, gout, jaundice, and wound healing in Iranian traditional medicine. Ajuga chamaecistus ssp. tomentella is an exclusive subspecies of Ajuga chamaecistus in the flora of Iran. Plants belong to Ajuga species are advantageous sources of phytoecdysteroids. 20-hydroxyecdysone (20E) is an important phytoecdysteroid with anabolic property. This study aimed to determine and quantify 20E in methanolic extract of aerial parts of Ajuga chamaecistus ssp. tomentella. The standard reference of 20E was isolated from n-butanolic fraction of aerial parts of Ajuga chamaecistus ssp. tomentella using high-performance liquid chromatography (HPLC). The analysis was carried out on an ODSA (C18) column with isocratic elution using water–acetonitrile (75:25). The standard calibration curve represented good linearity (r2 = 0.9997). The limit of quantification (S/N = 10) and detection (S/N = 3) were determined as 7.93  and  2.38  µg/ml,  respectively.  The  content  of  20E  in  methanolic  extract  of  Ajuga chamaecistus ssp. tomentella was determined to be 2.58% (w/w) (0.46% in dry plant). The quantitative proportion of the 20E found in Ajuga chamaecistus ssp. tomentella extract proposes the possible uses of this plant in commercial formulations or as a source of 20E.