This review describes the application of electrophoresis in a clinical laboratory to detect microheterogeneity of protein or sugar chain structure. We developed convenient method of isoelectric focusing using cellulose acetate membrane combined with various staining methods and improved transfer method with PVDF membrane. These methods enabled the easy analysis of clinical samples, and we have explored new biomarkers for various diseases. Target biomolecules of isoelectric focusing was as follows: sugar chains of α 1-acid glycoprotein and antichymotrypsin for inflammation, urinary transferrin for renal diseases, and sugar chains on M protein for multiple myeloma (MM). We revealed that sugar chains with highly reactive to Con A lectin was detected in the early stage of inflammation, while in the recovery phase of inflammation, sugar chains with highly reactive to DSA lectin increased. For isoelectric point of urinary transferrin, we found that transferrin with low isoelectric point increased in the early stage of nephropathy. Further, when glycosylation of M protein in the samples of MM patients with osteolysis was examined, sugar chains in the light chain on M protein showed high reactivity with Con A lectin. Thus, the analysis of protein binding sugar chains using isoelectric focusing is applicable to develop new biomarkers of various diseases, such as inflammation, nephropathy and cancer.