The by-product from tuna processing is a potential source of edible protein. Therefore, it is very important to extract protein from such raw materials for human food. In this study the optimum pH for protein extraction from tuna by-products was optimized by using isoelectric solubilization and precipitation processes. The Response Surface Methodology (RSM) and the single factor model were used for optimization of the protein extraction process. From ANOVA (one-factor design) tests, significant effects were detected for process variables, functional properties and stability between tuna protein isolate prototypes extracted at acidic and alkaline pH, the latter having the least Total Volatile Basic Nitrogen (TVB-N) and Thiobarbituric Acid Reactive Substances (TBARS), but the highest Water Holding Capacity (WHC), hardness, cohesiveness, springiness and viscosity values. The highest yield percentage was found for the alkaline aided process, too. The alkali-aided process recovered proteins of higher whiteness than the acid-aided process. Accordingly the optimum pH of protein extraction was obtained. The model was then validated and maximized based on the functional properties, stability and recovery yield data. Under the optimized pH, the experimental values were in good agreement with those predicted by the software. Then the properties of the optimum prototypes were compared to the fish protein isolated from different by-products. The results suggest that the proteins recovered from tuna processing by-products could be a valuable source of protein ingredient for fortification/ developing formulated ready-to-eat products.