Abstract: High-speed counter-current chromatography (HSCCC) was developed for separation of three high polarity flavonoid glycosides from EtOAc-soluble fractions of Hydrocotyle sibthorpioides Lam. The EtOAc-soluble fractions were subjected to silica gel column chromatography and further purification by Sephadex LH-20, eluted with gradient CHCl₃·MeOH (1:3) and MeOH·H₂O (7:3, V/V). After screening the solvent system, two phase solvent system consisting of n-hexane/n-BuOH/water/glacial acetic acid (1:1.7:1:0.1, V/V/V/V) were used to separation sample. In addition, the rotation speed, flow rate and detection wavelength were set at 2.0 mL/min, 800 r/min and 254 nm, respectively. The collected fractions were analyzed by HPLC and the purity were higher than 90%. Importantly, this three compounds were isolated from this plant for the first time. Their structures were identified by¹H NMR and¹³C NMR spectroscopy, named as myricetin 3, 3'-di-α-L-rhamnopyranoside, apigenin 7-O-β-D-glucuronide and luteolin 7-O-β-D-glucuronide. This work could provide supporting for study on the chemical constituent of Hydrocotyle sibthorpioides Lam.