Urine can help in diagnosis of different diseases including cancer and other patho-physiological conditions. Urine proteome studies have been mainly human- centric. No information is available on urinary proteome from bovine till date. In the present study, we have used 3 protein extraction methods such as ammonium sulphate precipitation, ProteoSpin column and diafiltration method from bovine urine for identification of urinary proteome. The tryptic peptides generated after In-gel and In-solution method were identified using LC/MS/MS (ESI-qTOF) which resulted in identification of 1582 proteins. In-gel trypsin digestion method revealed more protein (1191) in comparison to in-solution digestion method (541). Maximum proteins were identified in ammonium sulphate precipitation method (938) followed by ProteoSpin (606) and diafiltration (444) methods respectively. The profile of the identified proteins were compared with human urinary proteome of which 311 bovine urinary proteins matched with human. An exclusive list of 38 bovine urinary proteins with high protein scores were listed which are absent in human urine. All identified proteins were analyzed according to Gene Ontology which were classified according to cellular component, biological processes and molecular functions. This study reports for the first time an exclusive evidence of more than 1550 proteins in urine of healthy cow donors.