Many Eleutherococcus species grow in Siberia, China, Korea, Japan, and the Philippines. The most well known is Eleutherococcus senticosus, which contains pharmacologically active compounds, such as eleutherosides, flavonoids, vitamins, and complex polysaccharides. E. senticosus owes its medicinal properties mainly to eleutherosides. The objective of this study was to determine eleutherosides B, E, and E1 in the aerial parts of different Eleutherococcus species. The eleutherosides were extracted with ethanol, and the extracts were cleaned by SPE on C18 columns, and then analyzed by HPTLC. Silica gel plates with fluorescence indicator, designated F254, were used with chloroform-methanol-water (70 + 30 + 4, v/v/v) and chloroform-methanol-toluene-ammonium hydroxide (9 + 6 + 3 + 2, v/vv/v) mobile phases. Two-step elution with these mobile phases was used for the development of chromatograms. Eleutherosides were visualized by derivatization with Liebermann-Burchard reagent. This reagent was used for the first time to detect eleutherosides. Eleutherosides B, E, and E1 were detected in the fruits of the investigated species. E. senticosus contained three of the investigated compounds, and E. sessiliflorus, E. gracilistylus, and E. divaricatus two compounds each.