Gout is one of the most common forms of inflammatory arthritis, affecting over 8 million adults in the US. Individuals with gout are advised to avoid the habitual intake of purine-rich foods such as meats, seafood, purine-rich vegetables, and animal protein. An increased risk of developing or having subsequent attacks of gout is particularly associated with the consumption of seafood. However, clinical studies have shown that certain seafood and fish oil supplements that contain large amounts of omega-3 fatty acids provide important cardiovascular benefits. Individuals who might benefit from omega-3 fatty acid supplementation may therefore avoid fish oil products because they contain purines. Currently, there are no distinct high-performance liquid chromatography (HPLC) methods available in the literature that are validated as per the International Conference on Harmonisation (ICH) guidelines for the analysis of omega-3 fatty acid oils or fish oil containing products for purine content. A robust, fast, and efficient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the specific analysis of the naturally occurring purines guanine, purine, theobromine, and adenine. These purines are often found in fish oil and seafood. The analytical method reported herein quantifies all four purines in fish oil in about 20 minutes.