<p>Diabetes mellitus is a metabolic disease marked by chronic high blood levels of glucose (also known as hyperglycaemia), which over time can cause damage to the blood vasculature, leading to devastating complications. The increase in global prevalence and the number of cases of type 2 diabetes mellitus continues unabated, prompting diabetes mellitus researchers to continue studying the aetiology of the disease in search of ways to improve therapeutic outcomes. Over the years, numerous in vivo and in vitro mammalian models have been developed to investigate the mechanisms underlying diabetes mellitus and its complications. The customary use or induction of high levels of glucose in vivo or in vitro to recapitulate chronic hyperglycaemia in diabetes mellitus is central to these models. However, there has never been a consensus as to how to standardize the re-creation of glucotoxicity conditions so that the pathophysiological effects in human diabetes mellitus can be accurately mimicked experimentally. In this Review, we critically discuss glucotoxicity (without lipotoxicity) in diabetes mellitus and analyse the various in vivo and in vitro models used to study the conditions created by high levels of glucose in diabetes mellitus. Efforts to standardize experimental induction or administration of high levels of glucose, and understanding the pros and cons of each model, could help harmonize findings that will benefit the diabetes mellitus research community.</p>