The study of alkaloids from methanolic extract of Crucianella sintenisii from Rubiaceae family has not been studied yet. In this research, the methanolic extract was initially prepared from the aerial parts of Crucianella sintenisii, and then the resulting extract was acidified by acetic acid to remove neutral compounds. The neutral compounds were extracted by EtOAc (ethyl acetate). The resulting extract was alkalized with NH3 (25%), and then extracted with chloroform solvent. Finally, the crude alkaloid is extracted and ready to be injected into the MPLC device. One fraction was isolated, and the structure of 2-methyl-3-phenyl indole from the category of indole alkaloids was confirmed by using the spectroscopic techniques (NMR, ESI-MS, and IR). The therapeutic activities of the plant indole alkaloids are extensive, and most of these activities are related to the indole core in these structures.