AbstractEmulsion technology has been successfully applied to the fields of next‐generation high‐throughput sequencing, protein engineering and clinical diagnostics. Here, we extend its scope to proteomics research by developing and characterizing a method, termed iCLIP (in vitro compartmentalized linkage of interacting partners), which enables genes encoding interacting protein pairs to be linked in a single segment of DNA. This will facilitate archiving of the interactomes from library versus library two‐hybrid screens as libraries of linked DNAs. We further demonstrate the ability to interrogate a model yeast two‐hybrid iCLIP library for interactants by “PCR‐pulldown,” using a primer specific to a gene of interest along with a universal primer. iCLIP libraries may also be subjected to high‐throughput sequencing to generate interactome information. The applicability of the technique is also demonstrated in the related context of the bacterial two‐hybrid system.