Numerous ecdysteroids are isolated from the herb of Serratula wolffii Andrae, a cultivated plant. The isolation procedure includes a variety of low-pressure liquid chromatography, thin-layer chromatography (TLC), gel chromatography, and high-performance liquid chromatography (HPLC) methods. The progress of separation is monitored by TLC, and the final proof of purity is carried out by HPLC. The isolation process involves the removal of proteins, flavonoids, chlorophylls, other sterines, etc. The purification also includes the separation of the target ecdysteroids from each other. Isolation of the pure compounds requires 2-8 chromatographic steps. The consecutive steps are based on the different physicochemical properties of the ecdysteroids. In some cases, a special peak-cut method employing a flush of dichloromethane into the dichloromethane-isopropanol-water mobile phase is used. This flush of dichloromethane leads to an almost perfect separation of otherwise unresolved peaks. Two ecdysteroids, 25-hydroxydacryhainansterone and 14-epi-20-hydroxyecdysone, are identified as natural products for the first time. The structure-chiroptical relationships for some ecdysteroids are also discussed.