Nascent polypeptide chains emerging from the ribosome engage a range of co-translational factors at distinct phases of translation. These co-translational interactions are crucial for proper protein biogenesis and quality control pathways to maintain protein homeostasis. Hence, systematic identification of these co-translational interactors provides insights into how distinct polypeptide fates are determined. Here, we developed Nascent Chain Interactor Profiling (NCIP), a metabolic labelling and chemical crosslinking enabled proteomics method to identify proteins interacting with nascent polypeptide chains at a proteome-wide scale. Results from NCIP support a widespread mechanism of co-translational assembly of protein complexes and reveal TRIM25 as a novel co-translational E3 ubiquitin ligase. TRIM25 ubiquitinates misfolded nascent polypeptide chains for quality control at the ribosome. Our results provide a generalisable framework to systematically profile nascent polypeptide chains interactors.