A suitable combination of preparative scale separation methods results in effective clean-up of the ecdysteroids of Silene italica ssp. nemoralis (Waldst. and Kit.) Nyman. The isolation of minor ecdysteroids from the partially purified extract is based on the use of both droplet counter-current chromatography and low-pressure reversed-phase liquid chromatography. The purification is completed by preparative thin-layer chromatography and preparative high-performance liquid chromatography to obtain the minor ecdysteroids, such as 2-deoxy-20-hydroxyecdysone, shidasterone, 2-deoxy-polypodine B, makisterone C, and 9alpha,20-dihydroxyecdysone.