© 2022, Romanian Society for Pharmaceutical Sciences. All rights reserved.This study aimed to evaluate the antioxidant activity, the inhibitory effect on digestive enzymes linked to diabetes and the phytochemical composition of an active sub-extract obtained from Cousinia aintabensis (Asteraceae). The antioxidant effect of extracts was investigated with DPPH●, ABTS●+ and FRAP tests and the inhibition effect was tested on α-amylase and α-glucosidase. The quantitative and qualitative determination of the compounds within the active extract was carried out by LC-MS/MS. The highest total phenolic (185.816 ± 0.003 mg GAE/g extract) and total flavonoid content (147.423 ± 0.003 mg CA/g extract) was found in ethyl acetate sub-extract of C. aintabensis (CAE). It also showed the highest antioxidant activity on DPPH● (IC50 = 0.473 ± 0.056 mg/mL), ABTS●+ (at a conc. of 0.5 mg/mL equivalent to 0.543 ± 0.005 µM Trolox) and FRAP (at a conc. of 1 mg/mL equivalent to 2586 ± 6.421 mmol Fe2+) tests. Furthermore, the highest α-amylase and α-glucosidase activity were also found in CAE (IC50 = 0.361 ± 0.010 µg/mL for α-glucosidase and 1.010 ± 0.010 for α-amylase). According to LC-MS/MS analysis, the main compounds of CAE were cynarin and isorhamnetin 3-O-rutinoside.