Ecklonia stolonifera is a rich source of phlorotannins, which are responsible for the potent pharmacological effects associated with this seaweed. The purpose of this study was to develop a reversed-phase high-performance liquid chromatography method for the simultaneous determination of three major phlorotannins, eckol, dieckol, and phlorofucofuroeckol-A, in the extracts of Ecklonia stolonifera. The optimal chromatographic conditions were achieved on a Thermo Hypersil Gold C-18 column ($250{ imes}4.6;mm$ i.d., $5;{mu}m$) using linear gradient elution of acetonitrile and water containing 0.1% formic acid at UV 254 nm. The separated phlorotannins were identified by liquid chromatography-mass spectrometry. The high-performance liquid chromatography method showed good linearity ($r^2$ > 0.998), precision (1.4-9.5%), and accuracy (93.9-108.7%). The limits of detection ranged from 0.06 to $0.30{mu}g$/mL and the lower limits of quantitation ranged from 0.2 to $1.0{mu}g$/mL. Among phlorotannins, dieckol was the most abundant in both ethanol and ethyl acetate extracts of Ecklonia stolonifera.