Objective: To determine α-linolenic acid and linoleic acid in extract of Portulaca oleracea L. by high performance liquid chromatography (HPLC).Methods: The determination was done with a Shim-pack CLC-ODS (250 mm×4.6 mm, 5 μm) and a DIKMA Easyguard C18 (10 mm×4.6 mm). Elution was employed with the mobile phase of methanol-acetonitrile-0.5% phosphonic acid (60︰22︰18) at flow rate of 1.1 ml/min. Column temperature was 26 ℃. Detection wavelength was 210 nm. Injection volume was 25 μl.Results: The standard curves of α-linolenic acid and linoleic acid were linear in the range 0.016 2 to 0.194 4 mg/ml and 0.016 9 to 0.203 0 mg/ml, respectively. The regression equations were A=2.915 8×107 C+12 250.9, r=0.999 9 and A=1.366 4×107 C－9 759.39, r=0.999 9, respectively. The average recovery rates were 100.5 % and 100.8 %, respectively.Conclusion: The present method (HPLC) may be considered to be reliable and simple for the determination of α-linolenic acid and linoleic acid in extract of Portulaca oleracea L.