The Escherichia coli twin‐arginine translocation (Tat) system serves to export fully folded protein substrates across the bacterial cytoplasmic membrane. Respiratory [NiFe] hydrogenases are synthesised as precursors with twin‐arginine signal peptides and transported as large, cofactor‐containing, multi‐subunit complexes by the Tat system. Cofactor insertion and assembly of [NiFe] hydrogenases requires coordination of networks of accessory proteins. In this work we utilise a bacterial two‐hybrid assay to demonstrate protein–protein interactions between the uncharacterised chaperones HyaE and HybE with Tat signal peptide‐bearing hydrogenase precursors. It is proposed that the chaperones act at a ‘proofreading’ stage in hydrogenase assembly and police the protein transport pathway preventing premature targeting of Tat‐dependent hydrogenases.