Serratula centauroides L. is a plant species of Asteraceae family widespread in Cisbaiklia and containing phytoecdysteroids. As a result of chromatographic separation of the aerial part of S. centauroides growing in Buryatia Republic nine compounds were isolated and identified based on UV, NMR and mass spectrometry data. The presence of six compounds including isovitexirone, 24(28)-dehydromakisterone, 20-hydroxyecdysone 20,22-monoacetonide, 20-hydroxyecdysone 22-acetate, inokosterone and makisterone C was detected for the first time in this species. Three previously detected compounds were confirmed in S. centauroides like integristerone A, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone. According to HPLC data, the highest content of phytoecdysteroids was observed in the leaves of S. centauroides (20.64 mg/g) and lowest in tubular flowers (3.75 mg/g). The main component of the phytoecdysteroids sum was 20-hydroxyecdysone which concentration in the leaves reached 15.07 mg/g. Quantitative analysis of the aqueous medicinal preparations of S. centauroides (decoction, infusion) showed high phytoecdysteroids content (0.77–1.01 mg/mL) and its can be concluded as a source of this group of compounds.