The development, refinement and validation of in vitro digestibility assays for dietary protein and amino acids for single stomached mammals are reviewed. The general principles of in vitro digestibility assays and their limitations are discussed. In vitro protein digestibility assays must be accurate, rapid, cheap, simple, robust, adaptable and relevant to the processes of digestion, absorption, and metabolism. Simple in vitro methods have the potential to give useful measures of in vivo amino acid and protein digestibility for humans. In vitro methods, including the complex multi-component models of digestion simulating the various physical and chemical processes, require independent validation with in vivo data from the target species or an acceptable animal model using the most appropriate in vivo measure of digestibility. For protein sources devoid of anti-nutritional factors or plant fibre, true ileal digestibility is the recommended in vivo baseline, while for plant proteins the recommended in vivo assay is real ileal digestibility. More published comparative studies are required to adequately validate in vitro digestibility assays.