Glucose is an important nutrient and energy source in the body. However, too high concentration in the blood is harmful and may lead to several complications developing over time. It was estimated that 5 million people in the world died from complications related to diabetes during 2015. Several hormones and physiological factors are involved in the regulation of glucose homeostasis. To evaluate different aspects of glucose homeostasis and the effect of interventions, such as pharmacological treatment, glucose tolerance tests can be performed. In a glucose tolerance test glucose is administered either orally or intravenously, blood is sampled frequently and analyzed for different biomarkers. Mechanism-based pharmacometric models is a valuable tool in drug development, which can be applied to increase the knowledge about complex systems such as glucose homeostasis, quantify the effects of drugs, generate more information from clinical trials and contribute to more efficient study design. In this thesis, a new comprehensive mechanism-based pharmacometric model was developed. The model is capable of describing the most important aspects of glucose homeostasis during glucose tolerance test in healthy individuals and patients with type 2 diabetes, over a wide range of oral and intravenous glucose doses. Moreover, it can simultaneously describe regulation of gastric emptying and glucose absorption, regulation of the incretin hormones GLP-1 and GIP, hepatic extraction of insulin and the incretin effect, regulation of glucagon synthesis and regulation of endogenous glucose production. In addition, an interspecies scaling approach was developed by scaling a previously developed clinical glucose insulin model to describe intravenous glucose tolerance tests performed in mice, rats, dogs, pigs and monkeys. In conclusion, the developed mechanism-based models in this thesis increases the knowledge about short term regulation of glucose homeostasis and can be used to investigate combination treatments, drugs with multiple effects, and translation of drug effects between species, leading to improved drug development of new antidiabetic compounds.