The Sec61-associated Sec62/Sec63 complex supports translocation of only a subset of precursor polypeptides, i.e. in a substrate-specific manner. To characterize Sec62/Sec63-dependent precursors, we combined siRNA-mediated Sec62 or Sec63 depletion in Hela cells or CRISPR/Cas9-mediated knock-down in HEK 293 cells with label-free quantitative proteomics and differential expression analysis.