The rodent protection test (RPT) is used in antimicrobial research to confirm the in vivo efficacy of novel antibacterial, antiviral and antifungal agents. The RPT may involve lethal infection and thus has the potential to cause significant suffering. These Guidelines recommend refinements to the RPT which reduce animal use to a minimum and suggest cardinal clinical signs which act as predictors of lethality. Early recognition of these signs is imperative to minimize suffering, allow identification of humane end points and permit timely euthanasia. Constructive suggestions for the improvement of these Guidelines are welcomed.