This study was to investigate an analytical method for determining dieckol content in Ecklonia stolonifera extract. According to the guidelines of International Conference on Harmonization. Method validation was performed by measuring the specificity, linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ) of dieckol using high-performance liquid chromatography–photodiode array. The results showed that the correlation coefficient of calibration curve (R2) for dieckol was 0.9997. The LOD and LOQ for dieckol were 0.18 and 0.56 μg/mL, respectively. The intra- and inter-day precision values of dieckol were approximately 1.58-4.39% and 1.37-4.64%, respectively. Moreover, intra- and inter-day accuracies of dieckol were approximately 96.91- 102.33% and 98.41-105.71%, respectively. Thus, we successfully validated the analytical method for estimating dieckol content in E. stolonifera extract.