Abstract Lancea tibetica is an important traditional Tibetan medicinal plant that grows on the Qinghai-Tibet Plateau with great development potential in pharmaceutical industry. In this study, a combinative method using HPLC-DPPH and two-dimensional liquid chromatography has been developed to identify and separate antioxidants from Lancea tibetica. Under the target-guidance of HPLC-DPPH experiment, three antioxidant fractions from Lancea tibetica were recognized. Then, separation of the three fractions using two-dimensional semi-preparation liquid chromatography led to seven phenylpropanoids: (+)-pinoresinol-β-D-glucoside (1), isoacteoside (2), acteoside (3), tibeticoside (4),epipinoresinol (5), anthelminthicol (6) and phillygenol (7). As a result, seven major antioxidants in Lancea tibetica were isolated with more than 96% purity. Furthermore, in vitro bioassay against DPPH revealed compounds 1 – 7 with IC 50 values ranging from 6.16 ± 0.08 to 25.09 ± 0.11 (μM) and compounds 1 , 2 and 3 showed activities stronger than the two reference antioxidants (vitamin C, rutin), with IC 50 values of 6.16 ± 0.08, 8.93 ± 0.06 and 7.98 ± 0.05 (μM), respectively. Results of the present study indicated that the method was an efficient technique to systematically screen and isolate antioxidants from medicine crops. Graphical abstract Unlabelled Image Highlights • A novel screen and separation method for purification of antioxidants directly. • Seven antioxidants isolated from Lancea tibetica bioactivity-guided. • Isolated antioxidants with IC 50 values from 6.16 ± 0.08 to 25.09 ± 0.11 (μM). • The first report on antioxidant capacity of the compounds from Lancea tibetica.