Ultrasound-assisted11UAE – ultrasound-assisted extraction. (UAE), refluxing22RE – refluxing extraction. (RE), low pressure refluxing33LPRE – low pressure refluxing extraction. (LPRE) and Soxhlet44SE – Soxhlet extraction. (SE) extraction techniques were developed for determination of steroidal saponins content in Tribulus terrestris L. Extraction parameters for UAE (solvent base: methanol, ethanol, isopropanol and acetonitrile; the ratio of solvent to solid: 50–400 mL/g; isopropanol and acetonitrile concentration: 30–100%; extraction time: 15–120 min), RE (solvent base: isopropanol and acetonitrile; the ratio of solvent to solid: 50–400 mL/g; isopropanol concentration: 30–80%) and SE (extraction time: 4, 8, 12, 18 and 24 h) methods were optimized by single-factor experiment. Thermal decomposition of protodioscin was observed after 60 min of RE and 12 h of SE. Extraction time and temperature were optimized via dual-factor experiment for most promising RE and LPRE techniques. Comparison of UAE with RE, LPRE and SE methods was made for extraction of protodioscin and dioscin which are major polar and less polar T. terrestris saponins, correspondingly. The equal maximum of extraction yield was achieved using RE, LPRE and SE in optimized extraction conditions. Also, the possibility of complete isolation of target compounds at the first extraction step was shown in multi-step extraction experiment for RE and LPRE methods and reached saponin yields were also achieved after 12 h of SE. The accuracy for all studied extraction techniques was confirmed by spiking of the plant material with standards before extraction and HPLC-ESI–MS analysis.