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Tribulus is a well-known pharmaceutical herb that has been used for a long time in the traditional Chinese and Indian medicines for the treatment of various diseases. It has been found that plants belonging to the genus Tribulus are rich in biologically active furostane-, cholestane- and spirostane- type steroidal saponins. At the first step of this study several extraction techniques were applied in order to achieve comprehensive extraction of protodioscin and dioscin which are the major polar (furostane-type) and less polar (spirostane-type) constituents of Tribulus terrestris L. leaves with common diosgenin aglycone. For this purpose HPLS-ESI-MS/MS method was developed for determination of protodioscin and dioscin content in water-organic extracts. The optimal conditions were chosen for ultrasonic-assisted extraction in isopropanol and methanol based solvents. Then ultrasonic-assisted extraction in water-organic solvent was compared to maceration in 70% ethanol as a traditional method for preparation of T.terrestris infusion. It was shown that the same amount of protodioscin may be observed in ethanolic extract and water-methanol mixture after ultrasonic- assisted extraction, in which dioscin content was significantly lower.