The NeXtProt release 2018-09-17 reports 2,890 of proteins are remaining in missing proteins level, also known as PE 2,3,4 level (14.2%). Our goal is finding MS evidence of missing proteins and identification of biological roles in cells. First, we tried to find missing proteins with no MS evidences in Chromosome 9 using NeXtProt database. High obstacles acquiring MS evidences of missing proteins due to the very low expression level or specific expression pattern in certain cell types or tissues drive us to take advantage of overexpression system. In brief, we constructed 5 plasmids DNA (FOXD4, ARID3C, OR1J1, ANKRD18A, ZNF510) harboring cDNA of missing proteins fused to Halo Tag and artificially transfected into HEK293T cells. Overexpressed missing proteins were purified with Halo tag purification system and subjected to LC-MS/MS analysis. This strategy gave us great advance in detection of MS evidences for missing proteins. Using this method, we definitely get 2 MS evidences of missing protein (FOXD4, and ARID3C) under the rule of Human Proteome Project Data Interpretation Guidelines. In short, we used followed search parameter; peptide FDR < 0.01, protein FDR < 0.01, and 2 or more unique peptides with 7 amino acids. Additional cellular analysis identified that FOXD4 and ARID3C were localized in cytosol and nucleus respectively. In conclusion, overexpression and purification system we used in the study may be alternative method to identify MS evidences of missing proteins.