Two-dimensional gel electrophoresis (2-DE) is a widely used method for separation of the proteins of a proteome and it enables their detection in a large concentration range. Sample preparation for isoelectric focusing and SDS-PAGE electrophoresis as well as spot visualization determines the quality of the obtained protein maps. Computer analysis of the proteome maps allows comparison and detection differences in protein profiles. In combination with mass spectrometry (MS) it enables the identification of a single protein. Low-abundance proteins of physiological body fluids are considered as the potential source of diagnostic biomarkers. These are obtained by such techniques as affinity chromatography, immunoaffinity, and ultrafiltration. A combination of proteomic and metabonomic analysis provides a collection of new markers which are helpful in modern medical diagnostics. The combination of the 2-DE technique and 1H MRS enables monitoring mild cognitive impairment(MCI) and the evolution of Alzheimer disease (AD). Proteome analysis of the liver and red blood cells of patients with diagnosed schizophrenia indicates the importance of analyzing external tissue, not only cerebrospinal fluid, in the diagnosis of this disease. Proteomic techniques enable the identification of new biomarkers in rheumatic disease by analyzing plasma, articular fluid and tissues. New protein biomarkers (in plasma, serum, pancreatic juice, urine) enable earlier cancer diagnosis and disease monitoring. Proteome analysis of maternal serum and amniotic fluid creates the possibility detection of protein markers in prenatal tests diagnosing Down's syndrome. Proteomic studies enable assessment of the influence of environmental contamination on the immunological system.