AbstractpH‐zone‐refining counter‐current chromatography was successfully applied to the separation of alkaloids from a crude extract of Peganum harmala L. using a multilayer coil planet centrifuge. The experiment was performed with a two‐phase solvent system composed of methyl tert‐butyl ether/THF/water (2:2:3 by volume) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 1.2 g of the crude extract, 554 mg harmine and 325 mg harmaline were obtained each with a purity of over 96% as determined by HPLC. The structures of the isolated compounds were identified by electron ionization MS (EI‐MS), 1H NMR, and 13C NMR.