Ursolic acid (UA, 3 ?-hydroxy-urs-12-en-28-oic acid) are isomeric triterpenic acids. The high quantities of pentacyclic triterpenoids in Scabiosa species seems to be obvious and there is an evidence that most of pentacyclic triterpenoids that have been isolated are saponins. This is one of the most important characteristic of the genus Scabiosa, the main aglycones are ursolic acid and oleanolic acid. In the current study, isolation from the aerial part and roots of Scabiosa palaestina L. was performed using Preparative HPLC. Furthermore, detection and quantitation of ursolic acid was performed by high performance thin layer chromatography (HPTLC). The identification of isolated triterpenoid involves two methods including FT-IR coupled with LC-MS/MS that have been used for the simultaneous determination of the isolated UA. Quantitative analysis of Ursolic acid content in chloroform fractions revealed that both of the aerial parts and roots contain comparable concentration of 0.052 and 0.054 mg/ml respectively. The FT-IR and LC-MS/MS spectra of the isolated compound shows good agreement with those reported in literatures of Ursolic acid. Quantitative concentration of UA in chloroform fraction revealed that aerial parts and roots contain comparable concentrations and the spectral data for the isolated unknown were in good agreement with those reported in literature of UA.