Accurate quantification of low-abundance proteins in human serum and cell culture supernatant is crucial for biomarker discovery, disease diagnostics and bioreactor monitoring. However, the high dynamic range of protein concentrations in these complex biological fluids presents a significant analytical challenge. This work utilizes microporous glass-fiber membranes functionalized with affinity peptides to capture cardiac troponin I (cTnI) from human serum and erythropoietin (EPO) from Chinese hamster ovary (CHO) cell supernatant. Layer-by-layer adsorption of carboxylic acid-rich polyelectrolytes enables covalent functionalization of the membranes with cTnI- and EPO-specific peptides terminated with lysine residues. The affinity peptide–membrane system enables enrichment of cTnI in the ng/mL range while simultaneously depleting highly abundant proteins in diluted human serum. EPO-affinity membranes in 96-well plates allow near-real-time (<5 min) quantification of EPO in CHO cell supernatant through the binding of a secondary antibody conjugated to a fluorophore. These affinity peptide–functionalized, membrane-based platforms offer a simple and cost-effective strategy for quantifying low-abundance proteins.