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Summary: Here, we present a protocol for establishing a protein interactome based on close physical proximity to a target protein within live yeast cells. We describe steps for capturing both transient and stable binders by integrating a non-natural amino acid. We detail procedures for employing a site-directed method for labeling the surface that mediates protein associations and uncovers the binding sites on the interactors. Combined with mass spectrometry, our approach proves valuable in discovering binding partners and constructing a comprehensive protein-interaction network. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.