An efficient method for the screening and isolation of potent antioxidants from a tibetan medicinal plant by employing online HPLC-DPPH assay combined HSCCC has been successfully established. Six major constituents: boschnaloside (1), alyssonoside (2), leucosceptoside A (3), isoverbascoside (4), leucosceptoside B (5) and verbascoside (6) were isolated and purified from the water extract by HSCCC using ethyl acetate-nbutanol-water as two-phase solvent system. The results indicated that the combination of the online HPLC-DPPH assay with HSCCC could be suitable for the screening and separation of antioxidant compounds from extract of this plant or other medicine plants.