Two-dimensional electrophoresis results in an adequate resolution of the proteome of microorganisms to allow the detection and identification of specific antigens after blotting on membranes and overlaying the protein pattern with patient’s sera. The complement of all identified antigens presents the immunoproteome of a microorganism. All the antigens specific for a microorganism or even for a disease are identified by mass spectrometry. For identification, peptide mass fingerprinting is used, and post-translational modifications are detected by mass spectrometry MS/MS techniques. High-resolution two-dimensional electrophoresis and unambiguous identification are prerequisites for reliable results. After statistical analysis, the resulting antigens are candidates for diagnosis or vaccination and targets for therapy.