Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying: i) the length of the spacer arm (SA; 5-atoms, 7-atoms, and 14-atoms) linking the ligand to membrane surface; ii) the affinity ligand (pABA) density on membrane surface (5–25 nmoles per cm2). Resulting membranes were tested for their ability to bind plasminogen activators (PAs) from mono- and multi- component systems in batch mode. The membrane containing pABA linked through 7-atoms SA but similar ligand density as in the case of 5- or 14- atoms long SA was found to bind up to 1.6-times higher amounts of PA per nmole of immobilized ligand from conditioned HeLa cell culture media. However, membranes with similar ligand densities but different lengths of SA, showed comparable binding capacities in monocomponent system. In addition, the length of SA did not affect the selectivity of the ligand for PA. A clear inverse linear correlation was observed between ligand density and binding capacity until the point of PA binding optima was reached (11±1.0 nmoles per cm2) in mono- and multi- component systems for 7- as well as 14- atoms SA. Up to 200-fold purification was achieved in a single step separation of PA from HeLa conditioned media using these affinity membranes. The issues of ligand leaching and reuse of the membranes were also investigated. An extensive regeneration procedure allowed the preservation of approximately 95% of the PA binding capacity of the membranes even after five cycles of use.