Mass spectrometry-driven BLAST (MS BLAST) is a database search protocol for identifying unknown proteins by sequence similarity to homologous proteins available in a database. MS BLAST utilizes redundant, degenerate, and partially inaccurate peptide sequence data obtained by de novo interpretation of tandem mass spectra and has become a powerful tool in functional proteomic research. Using computational modeling, we evaluated the potential of MS BLAST for proteome-wide identification of unknown proteins. We determined how the success rate of protein identification depends on the full-length sequence identity between the queried protein and its closest homologue in a database. We also estimated phylogenetic distances between organisms under study and related reference organisms with completely sequenced genomes that allow substantial coverage of unknown proteomes.