Urine is a complex fluid, which is thought to contain valuable diagnostic information regarding general health. In particular, there is great diagnostic potential in the peptide and/or protein content of urine, but the information is present in low abundance. Most traditional proteomic techniques lack sufficient sensitivity/dynamic range, especially for dilute and/or complex samples. However, orthogonal separation methods can be applied prior to protein/peptide analysis to increase the success rate of urine proteomic studies and access this potentially valuable information. In this chapter, we describe isoelectric focusing (IEF) of intact urine proteins, via free flow electrophoresis (FFE), prior to typical peptide-based mass spectrometry analysis, facilitating the deep analysis of urine protein detection and identification, for biomarker discovery. Our work demonstrates that such an approach can be used as a preprocessing step and can be integrated into a workflow for the successful identification of protein components (biomarkers) from urine.