Sweeteners are used in the food industry to provide sweetness similar to sugar and to decrease the caloric intake and risks associated with obesity. However, some sweeteners are characterised by bitter, metallic and other off-tastes. Sensory and cellular studies have demonstrated synergies between sweetener blends, which are responsible for enhancing sweetness. This study aimed to identify new sweetener blends that are able to enhance sweetness intensity without causing bitter off-taste using in vitro functional expression of taste receptors. The dose-response of the sweet taste receptor (TAS1R2/TAS1R3) was determined for sucrose and 9 sweeteners and was consistent with their sweetness potency. Stimulation of TAS1R2/TAS1R3 by 6 binary sweetener blends confirmed 3 known synergies determined by sensory analysis, including sucralose/acesulfame-K, rebaudioside A/erythritol and rebaudioside A/thaumatin, and revealed 2 new synergies, known as, neotame/D-allulose and mogroside V/thaumatin. No synergy was observed for the rebaudioside M/mogroside V blend, probably due to their common binding sites on the sweet taste receptor. The ability of the 9 selected sweeteners to activate the 25 human bitter taste receptors (TAS2Rs) was tested. The cellular based assay demonstrated that sucralose, acesulfame-K, rebaudioside A, mogroside V and D-allulose activated at least 2 TAS2Rs. Sucralose, acesulfame-K and rebaudioside A exhibited lower EC50 values for TAS1R2/TAS1R3 than for TAS2Rs, which may explain their absence of bitter off-taste at low concentrations, unlike mogroside V and D-allulose. Our data provide a receptor-based understanding of the complex synergies among sweetener blends and an effective approach for testing new sweeteners while avoiding the activation of TAS2Rs.