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In the past few years, the interest in protein hydrolysates has been growing exponentially due to their nutritional and functional benefits for both human food and animal feed. In this context, total protein content is a key parameter for characterizing by-products, monitoring the hydrolysis processes, and the hydrolysates. This study aims to develop an accurate and expeditious, revisiting a merging zones flow-based spectrophotometric method for total protein quantification in food samples and hydrolysates. For this purpose, the conventional and reliable Biuret method was used. This method was chosen for its ease of preparation and the use of reagents with associated low toxicity. The flow-based approach consisted of a double injection of the sample and reagent, using a commutator, in a continuous stream, resulting in the reduction of the reagent consumption. For the development of the flow injection analysis (FIA) system, essential parameters, including reagent concentration, flow rate and reactor length, were optimized. The method was validated across various matrices, addressing potential interferences, and demonstrated repeatability and reproducibility. This optimized protocol offers a robust, efficient tool for the food industry, enhancing quality control and nutritional assessments.