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Abstract Phlorotannins are reported to have diverse biological properties. However, no analytical methods for the standardization of phlorotannin preparations have been reported. Herein, we developed and validated an analytical method for the determination of dieckol in phlorotannin extracts (PRT) using high-performance liquid chromatography (HPLC). The optimum HPLC conditions consisted of a Supelco Discovery C18 column stationary phase, a mobile phase (A: 15 % HPLC grade methanol in deionized water, B: methanol), UV detection at 230 nm, and a flow rate of 0.7 mL/min. The optimized chromatographic conditions were validated and exhibited good specificity and linearity (R 2 > 0.9994–1.0000). The recoveries were in the range of 100.9–102.3 %. The method had good intermediate (%RSD 1.2) and intra-day (%RSD 0.4–1.7) assay precisions. This HPLC method had good accuracy and quality in the determination of dieckol in PRT.