Biofouling on polymeric membranes poses a significant challenge in protein production and separation processes. We report here on the use of zwitterionic peptides composed of alternating lysine (K) and glutamic acid (E) residues to reduce biomolecular fouling on gold substrates and polymeric membranes within a protein production-mimicking environment. Our findings demonstrate that both gold chips and polymeric membranes functionalized with longer sequence zwitterionic peptides, along with a hydrophilic linker, exhibit superior antifouling performance across various protein-rich environments. Furthermore, increasing the grafting density of these peptides on substrates enhances their antifouling properties. We believe that this work sheds light on the antifouling capabilities of zwitterionic peptides in cell culture environments, advancing our understanding and paving the way for the development of zwitterionic peptide-based antifouling materials for polymeric membranes.