Various countercurrent chromatography (CCC) methods employ two-phase solvent systems for separation of flavonoids. CHCl3:CH3OH:H2O (4:x:2, for which x is between 2.5 and 4 for different samples) may be used for their prefractionation. Two-phase solvent systems can be divided into four types: CHCl3:CH3OH:H2O, CHCl3: CH3OH:n-BuOH:H2O, EtOAc:PrOH:H2O or BuOH: HOAc:H2O, and n-C6H14:EtOAc:CH3OH:H2O, respectively. CCC fractionation permits the use of either stepwise elution or gradient elution, and analytical-scale CCC can also be carried out for separation of flavonoids.