Whole cell protein and outer membrane protein (OMP) extracts were compared for their ability to differentiate and delineate the correct database organism to an experimental sample and for the degree of dissimilarity to the nearest neighbor database organism strains. These extracts were isolated from pathogenic and nonpathogenic strains of Yersinia pestis and Escherichia coli using ultracentrifugation and a sarkosyl extraction method followed by protein digestion and analysis using liquid chromatography tandem mass spectrometry (MS). Whole cell protein extracts contain many different types of proteins resident in an organism at a given phase in its growth cycle. OMPs, however, are often associated with virulence in Gram-negative pathogens and could prove to be model biomarkers for strain differentiation among bacteria. The mass spectra of bacterial peptides were searched, using the SEQUEST algorithm, against a constructed proteome database of microorganisms in order to determine the identity and number of unique peptides for each bacterial sample. Data analysis was performed with the in-house BACid software. It calculated the probabilities that a peptide sequence assignment to a product ion mass spectrum was correct and used accepted spectrum-to-sequence matches to generate a sequence-to-bacterium (STB) binary matrix of assignments. Validated peptide sequences, either present or absent in various strains (STB matrices), were visualized as assignment bitmaps and analyzed by the BACid module that used phylogenetic relationships among bacterial species as part of a decision tree process. The bacterial classification and identification algorithm used assignments of organisms to taxonomic groups (phylogenetic classification) based on an organized scheme that begins at the phylum level and follows through the class, order, family, genus, and species to the strain level. For both Gram-negative organisms, the number of unique distinguishing proteins arrived at by the whole cell method was less than that of the OMP method. However, the degree of differentiation measured in linkage distance units on a dendrogram with the OMP extract showed similar or significantly better separation than the whole cell protein extract method between the sample and correct database match compared to the next nearest neighbor. The nonpathogenic Y. pestis A1122 strain used does not have its genome available, and thus, data analysis resulted in an equal similarity index to the nonpathogenic 91001 and pathogenic Antiqua and Nepal 516 strains for both extraction methods. Pathogenic and nonpathogenic strains of E. coli were correctly identified with both protein extraction methods, and the pathogenic Y. pestis CO92 strain was correctly identified with the OMP procedure. Overall, proteomic MS proved useful in the analysis of unique protein assignments for strain differentiation of E. coli and Y. pestis. The power of bacterial protein capture by the whole cell protein and OMP extraction methods was highlighted by the data analysis techniques and revealed differentiation and similarities between the two protein extraction approaches for bacterial delineation capability.