The emerging formats of antibody fragments for biotherapeutics suffer from inadequate purification methods, delaying the advances of innovative therapies. One of the top therapeutic candidates, the single -chain variable fragment (scFv), requires the development of individual purification protocols dependent on the type of scFv. The available approaches that are based on selective affinity chromatography but do not involve the use of a purification tag, such as Protein L and Protein A chromatography, require acidic elution buffers. These elution conditions can cause the formation of aggregates and thereby greatly com-promise the yield, which can be a major problem for scFvs that are generally unstable molecules. Due to the costly and time-consuming production of biological drugs, like antibody fragments, we have en-gineered novel purification ligands that elute the scFvs in a calcium-dependent manner. The developed ligands are equipped with new, selective binding surfaces and were shown to efficiently elute all cap-tured scFv at neutral pH with the use of a calcium chelator. Further, two of three ligands were proven not to bind to the CDRs of the scFv, indicating potential for use as generic affinity ligands to a range of different scFvs. Multimerization and optimization of the most promising ligand led to a 3-fold increase in binding capacity for the hexamer compared to the monomer, in addition to highly selective and efficient purification of a scFv with > 95% purity in a single purification step. This calcium-dependent ligand could revolutionize the scFv industry, greatly facilitating the purification procedure and improving the quality of the final product.