The pathophysiology of diabetes as a disease is characterised by an inability to maintain normal glucose homeostasis. In type 1 diabetes, this is due to autoimmune destruction of the pancreatic β-cells and subsequent lack of insulin production, and in type 2 diabetes it is due to a combination of both insulin resistance and an inability of the β-cells to compensate adequately with increased insulin release. Animal models, in particular genetically modified mice, are increasingly being used to elucidate the mechanisms underlying both type 1 and type 2 diabetes, and as such the ability to study glucose homeostasis in vivo has become an essential tool. Several techniques exist for measuring different aspects of glucose tolerance and each of these methods has distinct advantages and disadvantages. Thus the appropriate methodology may vary from study to study depending on the desired end-points, the animal model, and other practical considerations. This review outlines the most commonly used techniques for assessing glucose tolerance in rodents and details the factors that should be taken into account in their use. Representative scenarios illustrating some of the practical considerations of designing in vivo experiments for the measurement of glucose homeostasis are also discussed.