Liquid Chromatography coupled to tandem mass spectrometry (nanoLC‐MS/MS) is a powerful analytical technique for the identification and mass analysis of complex protein mixtures. Here, we present a combination of methods developed for the extensive/deep proteomic analysis of purified ribosome/mRNA particles assembled in rabbit reticulocyte lysate (RRL). Ribosomes are assembled on chimeric biotinylated mRNA–DNA molecules immobilized on streptavidin‐coated beads and incubated with RRL to form initiation complexes. After washing steps, the complexes are trypsin‐digested directly on the beads in semi‐native condition or after their elution from the beads in denaturing Laemmli buffer. The nanoLC‐MS/MS analysis performed on complexes assembled on β‐globin, viral HCV, and histone H4 mRNAs revealed significant differences in initiation factors composition in agreement with models of translation initiation used by these different types of mRNAs. Using Laemmli‐denaturing condition induces release of deeply buried peptides from the ribosome and eukaryotic initiation factor 3 (eIF3) allowing the identification of the nearly complete set of ribosomal proteins.