We describe a simple method for isolation of human blood monocytes with the high purity required for proteomic analysis, which avoids contamination by other blood cells (platelets and lymphocytes) and the most abundant plasma proteins (albumin and immunoglobulins). Blood monocytes were purified by gradient centrifugation followed by positive selection with monoclonal antibodies coupled to paramagnetic beads. This method is compatible with flow cytometry, which was used to assess the purity of the cell population. After solubilization of monocytes, the proteins where analyzed by two-dimensional gel electrophoresis in several pH ranges. Image analysis of gels allowed the reproducible detection and quantification of the spots present in the gel. This method is useful for clinical studies of monocytes from a large number of patients, owing to its rapidity and reproducibility, which permits comparative analysis of normal vs pathological samples and allows follow up of the expressed proteins of monocytes from each patient.