We have developed a reliable genetic selection strategy for isolating interacting proteins based on the ‘hitchhiker' mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway. This method, designated FLI-TRAP (Functional Ligand-binding Identification by Tat-based Recognition of Associating Proteins), is based upon the Tat system's unique ability to efficiently co-translocate non-covalent complexes of two folded polypeptides. In the FLI-TRAP assay, one polypeptide is a fusion between an N-terminal Tat signal peptide (ssTorA) and the protein to be screened for interactions and the second is a fusion between a known or putative partner protein and mature TEM-1 β-lactamase (Bla). Using a series of c-Jun and c-Fos leucine zipper (JunLZ and FosLZ) variants of known affinities, we observed that only those chimeras that expressed well and interacted strongly in the cytoplasm were able to co-translocate Bla into the periplasm and confer β-lactam antibiotic resistance to cells. The utility of this assay was then demonstrated through: (1) random library selection of amino acid substitutions that restored heterodimerization to a non-interacting FosLZ variant; and (2) creation of artificial interaction interfaces between a Tat-targeted soluble scaffold (e.g., green fluorescent protein) and a cognate binding target fused to Bla. Since Tat substrates must be correctly folded prior to transport, FLI-TRAP favors the identification of soluble, non-aggregating, protease-resistant protein pairs and thus provides a powerful tool for routine selection of interacting partners (e.g., antibody-antigen) without the need for purification or immobilization of the binding target.