Urinary proteins provide valuable insights into renal health, with implications spanning human and domestic animal veterinary medical research. However, the field of marine mammal medicine lacks comprehensive studies on urine protein composition. This research aimed to fill this gap by 1) selecting an optimal search strategy that yields the highest number of protein families in the bottlenose dolphin urine based on post-translational modifications (PTMs), 2) describing the urine proteome of wild bottlenose dolphins (Tursiops truncatus) in the Gulf of the Mexico, USA, considering sex (females vs. males), site (Barataria Bay, LA vs. Sarasota Bay, FL), and comparing with California sea lions (Zalophus californianus). Ten urine samples (Barataria Bay, LA: N= 6; Sarasota Bay, FL: N=4) collected during 2023 catch-and-release heath assessment were proteolytically digested and analyzed using an UltiMate 3000 Nano LC and Fusion Lumos Orbitrap mass spectrometer. A 2-step search strategy, incorporating dehydroalanine formation and semi-trypsin on the list of unassigned spectra, significantly increased the number of identified protein families by an average of 6.2% compared to the 1-step strategy (P < 0.001, t = -8.32). The top 30 proteins in bottlenose dolphin urine were ranked according to an exponentially modified protein abundance index for comparison based on sex, site, and species. There were no significant differences in urine proteins between sexes or sites (padj > 0.05), although there was sperm contribution in two of the male bottlenose dolphin urine samples. Two putative antimicrobial proteins (cathelicidin and lysozyme) were identified and found to be abundant in bottlenose dolphin urine, similar to California sea lions. The study also identified 27 potential markers of acute kidney injury and 12 regulators of kidney stone formation. This study established a reference database of urinary proteins from bottlenose dolphins, aiding future research in monitoring and evaluating renal health in marine mammals.