The biochemical makeup of any organism provides insight into key factors regarding its biological functions. These factors can be explored using proteomics, which allows us to obtain a snapshot of the protein content and abundance in an organism, cell type or sub-cellular compartment. Here, we describe proteomic methodologies that can be used to dissect the biochemical mechanism of phytopathogenicity in oomycetes. These methodologies include protein extraction, purification, subsequent processing, mass spectrometry analysis, and qualitative and quantitative data processing of oomycete proteomes for comparative studies. Additionally, the use of mass spectra to assist in gene validation and modelling in unfinished oomycete genomes is also described.