With an increased focus on healthcare-associated infections, including multidrug-resistant pathogens, there is greater emphasis on environmental transmission and a need for methods to validate cleaning and disinfection programs in human and veterinary hospitals. This study, conducted at a university small animal teaching hospital, used a fluorescent tagging method to assess environmental cleaning practices across numerous surfaces and locations. Overall total cleaning success was only 50%, with surfaces categorized as primarily animal contact having a better cleaning rate (74.9%) than did surfaces designated as primarily human contact (41.7%). This low-cost method could be employed regularly to improve veterinary hospital cleaning practices.