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Leaves of Scorzonera incisa DC. (Asteraceae) are eaten raw against diabetes and headache. Evaluation of medicinal, edible plants using profiling methods has become a common phenomenon. Petroleum ether (PE), ethyl acetate (EA), and n‐butanol (BU) fractions were prepared from ethanol extract of the aerial parts of the plant. Isolation by HPLC and phenolic profiling by validated LC‐MS/MS method with 53 compounds were conducted on the EA fraction. GC‐MS method was used to profile the PE fraction. In‐vitro inhibitory activities of all fractions against α‐amylase, α‐glucosidase, AChE, BChE, tyrosinase and urease enzymes were measured. Isoorientin, vitexin, cynaroside and chlorogenic acid were isolated and characterized from the EA fraction as major compounds. LC‐MS/MS analysis quantified 36 compounds in the EA fraction. 20 compounds were tentatively determined in the PE fraction. Compounds with the same molecular and fragment ions, a low percentage of identified compounds, and very close polarities were revealed to be some limits of the nature for LC‐MS/MS profiling. Particularly, the presence and amount of substances other than the standard compounds used in the profiling methods and possible misdetections should not be neglected in evaluating medicinal plants and establishing a correlation between compounds and bioactivity. [ABSTRACT FROM AUTHOR]