Objective To determine the urinary polypeptide patterns of glomerulonephritis by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology. Methods Urinary samples of 29 healthy volunteers and 34 patients with glomerulonephritis, including 10 cases of IgA nephropathy (IgAN), 10 cases of membranous nephropathy (MN), 9 cases of minimal change disease (MCD) and 5 cases of lupus nephritis (LN), were collected and separated by magnetic bead,and were screened for polypeptide patterns with a novel high throughput method, MALDI-TOF MS. Results Under the relative molecular weights 10 000 Da, 85 protein peaks were detected in healthy controls group and 109 protein peaks were detected in glomerulonephritis group. Six peaks of 3371.5 Da, 4026.35 Da, 4085.32 Da, 4116.96 Da, 4126.32 Da and 9527.31 Da were up-regulated,while 8 peaks of 861.28 Da, 1205.41 Da, 1642.52 Da, 1913.15 Da, 1976.52 Da, 2087.74 Da, 2193.47 Da and 3015.57 Da were down-regulated by more than 2 folds (P<0.01) in glomerulonephritis group as compared to healthy controls. Urinary polypeptide patterns in different diseases differed significantly from each other, indicating specific disease pattern of polypeptide excretion. Conclusions MALDI-TOF MS is a fast, convenient and high throughput analyzing method capable of screening some relative specific, potential biomarkers from the urine of glomerulonephritis patients thus it possesses better clinical value.