Fish protein hydrolysates are obtained from fish waste streams via enzymatic processes. These hydrolysates contain bio-functional properties that can be used in nutraceuticals, supplements and food additives. The properties of the peptides usually relate to their molecular weight, amino acid composition, and amino acid sequence. To enrich specific bio-functionalities, separation units are required. Membrane separation is one of the down-streaming techniques for this purpose. In general, this technique separates molecules based on size. However, with charged peptides, the charge effect largely influences separation performance, including productivity and selectivity. Insight into the charge effect for complex hydrolysates is still rare. Therefore, this thesis investigated the charge effect on membrane performance when separating a fish protein hydrolysate. The charge effect depended on hydrolysate concentrations and the size difference between membrane pores and solutes. The proposed separation mechanism is helpful to design membrane processes.