Recent progress in mass spectrometry-based urinary proteomics highlights the potential of urine as a non-invasive source for disease biomarker discovery. Urine proteomics has advanced significantly over the past decade, with mass spectrometry enabling the identification of thousands of proteins in urine, including those from multiple organs. This review summarizes key developments in urinary proteomics, emphasizing its applications in diagnosing and monitoring various diseases, including cancer, diabetes, and chronic kidney disease. Urine is a valuable biospecimen due to its non-invasive collection and wide availability. However, factors such as sample collection methods, protein normalization, and inter-individual variability can affect the accuracy of urinary proteomic analysis. Techniques like MALDI-TOF-MS, CE-MS, and LC-MS/MS have been employed to profile urine proteins, with LC-MS/MS being the most commonly used method. These techniques allow for the identification of both abundant and low-abundance proteins, providing insights into disease mechanisms and potential biomarkers. Urinary proteomics has been applied to various conditions, including bladder cancer and diabetic nephropathy, where specific proteins and peptides have been identified as potential biomarkers. Glycoproteomics and peptidomics have also been explored, revealing the importance of glycosylation and peptide profiles in disease diagnosis. Despite its promise, challenges such as variability in protein abundance and the need for standardized protocols remain. Overall, urinary proteomics offers a non-invasive approach for biomarker discovery and disease monitoring, with significant potential for future clinical applications.Recent progress in mass spectrometry-based urinary proteomics highlights the potential of urine as a non-invasive source for disease biomarker discovery. Urine proteomics has advanced significantly over the past decade, with mass spectrometry enabling the identification of thousands of proteins in urine, including those from multiple organs. This review summarizes key developments in urinary proteomics, emphasizing its applications in diagnosing and monitoring various diseases, including cancer, diabetes, and chronic kidney disease. Urine is a valuable biospecimen due to its non-invasive collection and wide availability. However, factors such as sample collection methods, protein normalization, and inter-individual variability can affect the accuracy of urinary proteomic analysis. Techniques like MALDI-TOF-MS, CE-MS, and LC-MS/MS have been employed to profile urine proteins, with LC-MS/MS being the most commonly used method. These techniques allow for the identification of both abundant and low-abundance proteins, providing insights into disease mechanisms and potential biomarkers. Urinary proteomics has been applied to various conditions, including bladder cancer and diabetic nephropathy, where specific proteins and peptides have been identified as potential biomarkers. Glycoproteomics and peptidomics have also been explored, revealing the importance of glycosylation and peptide profiles in disease diagnosis. Despite its promise, challenges such as variability in protein abundance and the need for standardized protocols remain. Overall, urinary proteomics offers a non-invasive approach for biomarker discovery and disease monitoring, with significant potential for future clinical applications.