Veterinary medicines are subject to a rigorous evaluation with regard to safety, efficacy and quality before they are licensed. For drugs used in food producing animals, it is necessary to establish what is referred to as the acceptable daily intake (ADI), this is defined as an estimate of the amount of a substance, expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable risk to human health. It is necessary to determine a toxicological, pharmacological and microbiological ADI. This article describes a recently harmonized guideline that outlines the process for determining the need for a microbiological ADI and discusses the test systems that take into account the complexity of the human intestinal flora. The described process is used to address the effects of antimicrobial drug residues on human intestinal flora for regulatory purposes. The guideline does not recommend any one particular system for use in regulatory decision making but provides recommendations for a harmonized approach to establish a microbiological ADI and offers test options rather than specifying a testing regimen. The process and the challenges of this new guideline are discussed.