OBJECTIVE: To optimize a pretreatment method of urine proteomics in children with primary nephrotic syndrome. METHODS: Urine from children with primary nephrotic syndrome was treated in different pH and isolated by cold acetone precipitation for different durations. Then the amounts and kinds of proteins were compared by quantify, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) in order to optimize a way to deal with urine protein. RESULTS: Most proteins were obtained at pH 2.7. The amounts of protein precipitated by acetone for 0.5 hr was obviously less than those precipitated for 1 and 2 hrs (P<0.05), while there was no significant difference between the amount of protein precipitated for 1 and for 2 hrs. Protein precipitated by cold acetone for 1 hr at pH 2.7 was selected as the best pretreatment method. Satisfactory 2-DE maps can be acquired. CONCLUSIONS: Urine protein can be best obtained at pH 2.7 and precipitated by cold acetone for 1 hr.