Background and Objective: The opportunistic pathogen Pseudomonas aeruginosa secrets a capsule-like polysaccharide called alginate which is important for evasion of host defenses, especially in patients with suppressed immunity. Method of alginate determination has an important role in the study of microbial alginate. In this study, a novel method for alginate determination by highperformance liquid chromatography (HPLC) was introduced. Materials and Methods: Standard alginate was used for construction of standard curve and standard mucoid and non-mucoid strains of Pseudomonas aeruginosa were used as positive and negative samples respectively. The method of Toyoda was modified for determination of microbial alginate. HPLC determination was performed using a Resolve C18 column (3.9 × 150 mm, Waters, Milford, MA) and acetonitrile-water-butyl acetate (55: 42: 3) as the mobile phase at a flow rate of 0.6 ml/min and detection at 565 nm. Results: The obtained data indicated that minimal detectable concentration of alginate by this method is 20 μg/ml. The method was linear over the range of 1-1000 μg/ml of alginate. The retention time was about 10 min. Conclusion: The proposed method was used for determination of alginate in standard mucoid and non-mucoid strains of Pseudomonas aeruginosa. The results of this study showed that the proposed method is a simple and valid method for bacterial alginate assay.