The administration of antimicrobial agents to livestock creates potential for antibiotic residues to enter the food supply and be consumed by humans. Therefore, as a process of food animal drug registration, national regulatory agencies and international committees evaluate data regarding the chemical, microbiologic, pharmacokinetic, pharmacodynamic, pharmacologic, toxicologic, and antimicrobial properties of veterinary drugs to assess the safety of ingested antimicrobial residues to consumers. Currently, European, Australian and United States guidelines for veterinary drug registration require a safety assessment of microbiologic hazards from consumption of antimicrobial residues taking into account the potentially adverse effects on human intestinal microflora. The main concerns addressed are selection of resistant bacteria in the gastrointestinal tract and disruption of the colonization barrier of the resident intestinal microflora. Current requirements differ among national agencies. Efforts are ongoing internationally to review and harmonize approaches and test methods and protocols for application to these microbiologic safety evaluations of antimicrobial drug residues in food. This review describes the background to current regulatory approaches used in applying in vitro and in vivo methods to set a microbiologic acceptable daily intake for residues in food derived from animals treated with an antimicrobial agent. This paper also examines the current research needs to support these evaluations.