This work describes the assessment of a de novo synthetic affinity ligand for recombinant human erythropoietin (rHuEPO), based on the multicomponent Ugi reaction. Four Ugi ligands were designed based on the X-ray crystallographic structure of the complex between human erythropoietin and site 1 of its cell-surface receptor (EPObp)(2); screening of the ligands with pure rHuEPO samples identified a lead ligand (A9C10I8) immobilized on aldehyde-functionalized agarose beads, which was able to bind and elute erythropoietin, as determined by SDS-PAGE and Western blot analyses. Furthermore, small-scale affinity chromatography performed on the immobilized adsorbent showed its ability to isolate rHuEPO from a spiked mammalian cell supernatant with a purity of approximate to 80%, as estimated with gel densitometry. This approach could lead to the development of a cost-effective downstream process for rHuEPO, as an alternative to the current multi-step purification protocols. Biotechnol. Bioeng. 2013;110: 3063-3069. (c) 2013 Wiley Periodicals, Inc.