Retro-translocation from the ER to the cytosol of proteins within the secretory pathway takes place on misfolded molecules that are targeted for degradation by the cytosolically located 26S proteasome complex. Retro-translocation occurs also for other proteins (such as calreticulin) that, despite being synthesized and transported to the ER, are in part dislocated to the cytosol. We have taken advantage of the E. coli derived biotin-ligase (BirA) expressed in the cytosol of mammalian cells to specifically biotin-label in vivo proteins within the secretory pathway that undergo retro-translocation. We validated the method using four different proteins that are known to undergo retro-translocation upon different conditions: the human trans-membrane protein MHC class-I α chain (MHC-Iα), the Null Hong Kong mutant of the secretory α1 anti-trypsin (NHK-α1AT), the immunoglobulin heavy chain (HC) and the ER chaperone calreticulin (Crt). We observed specific mono-biotinylation of cytosolically dislocated molecules, resulting in a novel, reliable way of determining the extent of retro-translocation.