Consumption of non-nutritive sweeteners (NNS) is often advocated as a strategy to maintain a palatable diet without the metabolic detriments of consuming added sugar. There is conflict however, regarding the safety of NNS consumption. Epidemiology data has reported potential increases in cardiometabolic risk in humans with supporting evidence from basic science studies of rodents. We hypothesised that a key source of this conflict is the use of NNS models in basic science that lack ecological validity. We report here, a prospective audit of over 12 000 products in the Australian food supply. The primary aim of this study was to investigate the prevalence of NNS and low energy sweeteners (LES). Ingredients lists of all food products, oral hygiene products, vitamins and medicines available for sale in a single Woolworths store were photographed in September of 2018. Lists were reviewed to identify all NNS and LES present. 1034 products contained either an NNS or LES or a combination of both. The most common NNS used in food products were steviol glycosides (n = 183), sucralose (n = 172), acelsulfame-K (n = 159) and aspartame (n = 96). Of the 442 food products containing a NNS, 173 of these products contained more than one NNS across 18 unique combinations. There were distinct NNS prevalence patterns across food categories. For example, Acesulfame-K and Aspartame were the most commonly used NNS in gums, mints, and soft drinks. Sucralose and steviol glycosides were the most commonly used sweeteners in protein bars. While saccharin appeared in only 2 food products, but 148 oral hygiene products. This audit demonstrates the complex patterns of sweeteners that individuals may consume in Australia and presents the challenges for basic science in designing models with high ecological validity to inform clinical practice and public health policies aimed at reducing sugar consumption.