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Summary: Here, we provide a protocol for the systematic screening of protein-protein interactions mediated by short linear motifs using the Protein Interaction Screen on a peptide Matrix (PrISMa) technique. We describe how to pull down interacting proteins in a parallelized manner and identify them by mass spectrometry. Finally, we describe a bioinformatic workflow necessary to identify highly probable interaction partners in the large-scale dataset. We describe the application of this method for the transient interactome of the claudin protein family.For complete details on the use and execution of this protocol, please refer to Suarez-Artiles et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.