An improved procedure by using ureagradiont gel for protein folding and unfolding investigation is provided. Some key techniques such as urea gradient homogeneity, gel viscosity and gel edge effects have been discussed. An inverse gradient of glycerol is used to compensate for the small effects of urea on the electrophoretic mobilities of both folded and unfolded proteins. The glycerol gradient used here of 15% at 0mol/L urea to 0% at 8 mol/L urea was designed to give a constant mobility of creatine kinase at all urea concentrations. The gel was photopolymerized in the presence of riboflavin. 5% stacking gel and sample combs were also used on urea gradient gel to improve the sample pattern.