Abstract: Background and Objective: Study evaluates the suitable chemical method for the determination of the freshness of refrigerated seafood. Hypoxanthine content (Hx) of the refrigerated seafood was analysed and it was compared with the level of volatile amines production due to bacterial spoilage. Materials and Methods: Fresh seafood such as Stolephorus commersonii, Scomberomorus koreanus and Portunus sanguinolentus were collected from the fishing harbour of Tuticorin. The fishes were analyzed initially for TMA-N, TVB-N, hypoxanthine and pH content and it was stored at -4°C for 30 days. Sampling was done for every 10 days interval and it was checked for all the above quality indicators. Results: The results showed that volatile amines (TVB-N) and (TMA-N) were not found in stored seafood till the end of 10 days storage. Changes in pH and hypoxanthine content were noticed at the initial storage period itself. Refrigeration of sea foods are common at homes and at this condition even the superior quality stored fish has hypoxanthine formation and it gradually increase during the storage period. Hx assay reflects ATP break down and ATP-related compounds and the degradation of ATP to Hx has been attributed to muscle endogenous autolytic enzymes. The enzymatic reaction occurs in the initial stage of storage at low temperature but the contents of trimethylamine-nitrogen and total volatile base-nitrogen increase progressively during the later period of spoilage. Thus, hypoxanthine is regarded as the major catabolite of adenosine triphosphate while it was formed in refrigerated situation and it is a valuable freshness marker in stored fish. Conclusion: Thus, the study reveals that Hx measurements have some advantage over TMA-N and TVB-N analyses and Hx could be used as a superior spoilage indicator in the refrigerated sea foods. This study proves that the Hx could be used as a spoilage indicator and the sea foods stored at -4°C, should be consumed within 10 days of storage period to avoid Hx accumulation.