Cleaning and disinfection are critical areas of Veterinary hospital biosecurity. Without a validated biosecurity program, veterinary hospitals are in a vulnerable state. Potential outbreaks of pathogenic microorganisms could occur causing loss of: patient lives, hospital revenue and hospital prestige. Instances of pathogenic outbreaks have been recorded in small animal settings (i.e. veterinary clinics and shelters) and in large animal settings (i.e. farms and hospitals.) Veterinary hospitals, often considered the gold standard of veterinary care are not immune to biosecurity breaches. In last 5 years 82% of veterinary teaching hospitals reported nosocomial infections (Anderson, 2010). The aim of this project was to validate cleaning and sanitation procedures that are already in place in the Harry T. Peters, Jr. Large Animal Hospital located in Blacksburg, VA. This project compared two sanitation protocols and validated them using environmental sampling. Using chi-square analysis results indicated there was no significant difference between the sanitation methods.