The biological screening of the various Ruta chalepensis L. extracts through several in vitro antioxidant assays, including DPPH radical scavenging, scavenging activity of ABTS radical cation, reducing power and quantification of the total antioxidant capacity identified the MeOH60 fraction (F6) as having antioxidant properties, suggesting strong therapeutic potential. This fraction was therefore chosen for the structural identification of chemical compounds composing it. 2D NMR analyses allowed for the complete characterization of its primary compound, which was identified as nearly pure rutin. Rutin was isolated as a yellow powder, whose molecular formula C27H30O16 was deduced through the analysis of mass spectrometry data (m/z = 611 [M+H]+). This compound exhibits two adsorption maxima at ?max = 264 and 353 nm. HPLC, IR and UV data further confirm that rutin is the predominant component in fraction 6.