Data on urine proteome in big cats are lacking, therefore the aim of our research is to study urine proteome with sensitive analytical methods to monitor the health status in Panthera leo, Panthera tigris and Panthera pardus. Urine samples were collected 4 times from May to December 2012, by noninvasive methods from 2 tigers, 2 lions and 3 leopards from Rome’s bioparco. Urinary total proteins (UTP), creatinine and the urinary protein to creatinine ratio (UP/C) were measured by automated methods (Olympus AU400) routinely used for dogs and cats. Urine samples were analyzed by sodium-dodecyl-sulphate poliacrilamide gel electrophoresis (SDS-PAGE, Invitrogen®) to separate urinary proteins. Mean UTP values were not significantly different in lions, tigers and leopards (40, 43, 46 mg/dl respectively), while mean UP/C values were significantly lower in lions (0.11) than in tigers (0.28) but not than leopards (0.22). In absence of reference ranges, these values could be considered as representative of healthy animals. Regarding qualitative proteinuria, with the highly sensitive SDS-PAGE, 29 different protein bands were visualized in lions, 26 in tigers and 30 in leopards, including the 2 most abundant ones, which could be identified as albumin and putative cauxin, a specific feline’s protein. Noninvasive sampling was easy to apply without causing stress to animals and allowed to collect valuable samples for routine and/or more sensitive analysis.