Objectives It is important to use a sensitive method to detect monoclonal protein (MP) because asymptomatic patients with MP develop multiple myeloma or similar myeloproliferative disorder at the rate of 1-2% a year. The aim of this study was with comparative analyzes and interpretations of results immunofixation electrophoresis (IFE) and immunosubtraction electophoresis (ISE) determine the advantages and disadvantages of ISE and also assess whether this method can replace IFE in the detection and immunotyping of MP in the near future. Patients and Methods Processed are 50 serum patient samples from the Department of Hematology, Clinic for Internal Medicine, University Hospital Centre Zagreb. Patients are selected due to suspicion of monoclonal gammopathy or a condition characterized by the presence of MP. Serum protein electrophoresis was perform in all samples by capillary zone electrophoresis (CZE) method using Capillarys 2 Sebia system. Concentration of immunoglobulin G, A and M was measured on Roche Cobas 6000cee by turbidimetric procedure. IFE was perform on Sebia Hydrasys using Hydragel 2/4 IF gels. ISE was perform on Capillarys2. IFE is based on detecting appearance of monoclonal band after agarose gel electrophoresis and immunoprecipitation. The principle of ISE involves comparision of electrophoretic reference pattern with specific electrophoretic pattern obtained after immunoreaction with specific antibody bound to Sepharose beads. Results In 44/50 samples obtained results were equal and not diffucult to interpret by both methods. In the remaining 6 samples (biclonal gammopathy and reserved polyclonal synthesis of uninvolved immunoglobulin) the obtained results by IFE were easier to describe than obtained by ISE which can be explained by greater experience in interpreting immunofixation results. Conclusion The ISE is a good alternative for MP immunotyping in clinical laboratory. The method is fast, fully automated, but comparing to IFE lower sensitivity in detecting MP. Therefore, experienced and well trained laboratory professionals are necessary to interpret results of ISE .