Urine is a non-invasive source of biological fluid, which reflects the physiological status of the mammals. We have profiled the cow urinary proteome and analyzed its functional significance. The urine collected from three healthy cows was concentrated by diafiltration (DF) followed by protein extraction using three methods, namely methanol, acetone, and ammonium sulphate (AS) precipitation and Proteo Spin urine concentration kit (PS). The quality of the protein was assessed by two-dimensional gel electrophoresis (2DE). In-gel digestion method revealed more proteins (1191) in comparison to in-solution digestion method (541). Collectively, 938, 606 and 444 proteins were identified in LC-MS/MS after in-gel and in-solution tryptic digestion of proteins prepared by AS, PS and DF methods, respectively resulting in identification of a total of 1564 proteins. Gene ontology (GO) using Panther7.0 grouped the majority of the proteins into cytoplasmic (location), catalytic activity (function), and metabolism (biological processes), while Cytoscape grouped proteins into complement and coagulation cascades; protease inhibitor activity and wound healing. Functional significance of few selected proteins seems to play important role in their physiology. Comparative analysis with human urine revealed 315 overlapping proteins. This study reports for the first time evidence of more than 1550 proteins in urine of healthy cow donors. This article is part of a Special Issue entitled: Proteomics in India.