Interrogation of the urinary proteome for clinically useful biomarkers of disease will require normalization of methods for protein extraction and sample handling. Variations in collection methods and other procedures may introduce significant discrepancies in qualitative and quantitative measurements. Here we demonstrate that the method of protein extraction, length of handling at room temperature, and repetitive freeze-thaw cycles do not seem to alter the urinary proteome at either the protein or peptide level in a manner that degrades information obtainable by mass spectrometry.