Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners by mass spectrometry. Since contamination by streptavidin limits protein identification, we here present protease-resistant streptavidin beads (prS). Their application to different biotinylated biomolecules (DNA, protein and lipid) result in >100 streptavidin contamination and better coverage of both the target and its protein interactors without the need for sample pre-fractionation.