There is at least one additional targeting pathway for proteins to the human endoplasmic reticulum, which involves a cytosolic as well as an ER-membrane resident receptor, belonging to the family of PEX proteins. In contrast to the precursor protein targeting pathways, the additional pathway targets proteins to distinct ER sites and does not target proteins to the Sec61 complex. To characterize substrate proteins of this pathway, we combined siRNA-mediated PEX3-depletion in HeLa cells with label-free quantitative proteomics and and differential protein abundance analysis.