Antibody fragments and their engineered variants show true potential as next-generation therapeutics as they combine excellent targeting with superior biodistribution and blood clearance. Unlike full antibodies, however, antibody fragments do not yet have a standard platform purification process for large-scale production. Short peptide ligands are viable alternatives to protein ligands in affinity chromatography. In this work, an integrated computational and experimental scheme is described to de novo design 9-mer peptides that bind to Fab fragments. The first cohort of designed sequences was tested experimentally using human polyclonal Fab, and the top performing sequence was selected as a prototype for a subsequent round of ligand refinement in silico. The resulting peptides were conjugated to chromatographic resins and evaluated via equilibrium and dynamic binding studies using human Fab-κ and Fab-λ. The equilibrium studies returned values of binding capacities up to 32 mg of Fab per mL of resin with mild affinity (KD ∼ 10-5 M) that are conducive to high product capture and recovery. Dynamic studies returned values of product yield up to ∼90%. Preliminary purification studies provided purities of 83-93% and yields of 11-89%. These results lay the groundwork for future development of these ligands towards biomanufacturing translation.