Single-chain variable fragments (scFvs) specific for tumor-associated cell surface antigens are the most broadly used reagents to direct therapeutic or diagnostic effector molecules, such as toxins, radioisotopes, and CD3-stimulating scFvs, to tumors. One novel class of effector molecules that can be targeted to tumors by scFvs are ligands of the tumor necrosis factor (TNF) family. Typically, these molecules have apoptosis inducing and/or immune stimulating properties and are therefore highly attractive for cancer treatment. N-terminal fusion of scFvs does not interfere with the receptor binding capabilities of TNF ligands and thus allows the straightforward generation of scFv TNF ligand fusion proteins. We report here a protocol for the purification of eukaryotically produced scFv TNF ligand fusion proteins based on affinity chromatography on anti-Flag agarose and further describe assays for the determination of the targeting index of this type of scFv-targeted proteins.