Abstract: Some salt and denaturants suppress the sample signals in the determination of protein molecular weight by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), producing the ion peaks that is difficult to be explained, thus the additives in samples should be removed before measurement. A new method for on-line purification of samples with MALDI-TOFMS was established. The standard lysozyme was treated into 100 mmol/L Tris-HCL solution containing 6 mol/L guanidine chloride and 2% SDS surfactant with nitrocellubse membrane as stationary phase. The results showed the method is simple, rapid. Meanwhile, the intensity of signal and the sensitivity were obviously enhanced.