Updated project metadata. Mitochondria are complex organelles with different compartments, each harboring their own protein quality control factors. While the chaperones of the mitochondrial matrix have been well characterized, it is poorly understood which chaperones protect the mitochondrial intermembrane space. We show that cytosolic small heat shock proteins are imported under basal conditions into the mitochondrial intermembrane space, where they operate as molecular chaperones. To identify chaperone substrates with MS/MS, we used a molecular trap variant of HSPB1/Hsp27 (S135F mutant). Through affinity-enrichment co-immunoprecipitation we identified significantly enriched substrates versus GFP-negative control.