Teixeira, RaquelRibas, Tânia C. F.Almeida, AndréPintado, ManuelaRangel, António O. S. S.2026-04-212026-04-212026-06-01a6d223d0-9420-429c-a27f-bf5368a13ecdhttp://hdl.handle.net/10400.14/57541Protein hydrolysates are increasingly used in feed, underscoring the need for analytical tools for the rapid, reliable determination of total protein. This study revisited a merging-zones flow-based spectrophotometric method for minimizing reagent consumption and employs the Biuret reaction for the quantification of total soluble protein in by-products hydrolysates. The analytical method was optimized across different physical and chemical parameters such as: flowrate, reactor length, sample injection volume and reagents concentration. The use of different matrices relevant in the hydrolysis processes (acetate, phosphate, and hydrochloric acid) showed no significant interference (<10%) on the method's performance. Under optimal conditions, the method quantified hydrolysate protein within a dynamic range of 0.100–2.00 mg mL⁻¹ , with LOD and LOQ of 0.069 and 0.290 mg mL⁻¹ , respectively. Analyses of hydrolysates showed no significant differences compared with the reference method (<10%) while reducing analysis time from 30 min to 3 min per sample (triplicate). The method’s greenness, assessed by AGREE software, showed an improved score, from 0.58 to 0.76. The optimized protocol provides a fast, robust, and greener approach for monitoring protein hydrolysates in the food industry.engProtein quantificationHydrolysatesBiuret methodFlow Injection AnalysisSpectrophotometryresearch article10.1016/j.jfca.2026.109178105036184226001756278500001