Historically many genome annotation strategies have lacked experimental evidence at the protein level, which and have instead relied heavily on ab initio gene prediction tools, which consequently resulted in many incorrectly annotated genomic sequences. Proteogenomics aims to address these issues using mass spectrometry (MS)-based proteomics, genomic mapping, and providing statistical signi fi cance measures such as false discovery rates (FDRs) to validate the mapped peptides. Presented here is a tool capable of meeting this goal, the UCSD proteogenomic pipeline, which maps peptide-spectrum matches (PSMs) to the genome using the Inspect MS/MS database search tool and assigns a statistical signi fi cance to the match using a target-decoy search approach to assign estimated FDRs. This pipeline also provides the option of using a more reliable approach to proteogenomics by determining the precise false-positive rates (FPRs) and p -values of each PSM by calculating their spectral probabilities and rescoring each PSM accordingly. In addition to the protein prediction challenges in the rapidly growing number of sequenced plant genomes, it is dif fi cult to extract high-quality protein samples from many plant species. For that reason, this chapter contains methods for protein extraction and trypsin digestion that reliably produce samples suitable for proteogenomic analysis.