Nutritional supplements are used to strengthen deficiencies and rebuild the proper functions of the body. They contain two main classes of chemical compounds: excipients including bulking agents, sweeteners, acidity regulators, antifoam agents, and active molecules such as vitamins and minerals. Vitamins and minerals are known to generate bitterness, which may contribute to off-taste of nutritional supplements, which may lead to the reduction of their intake and decrease of the treatment’s efficiency. To better understand the mechanisms involved in off-taste perception, we combined human sensory analysis and taste receptor functional assays. In order to identify and quantify the nature of these negative perceptual sensations, 16 trained panelists carried out a sensory characterization of some effervescent nutritional supplements using the quantitative descriptive analysis method. We thus demonstrated that the bitterness is the main taste quality responsible for the off-taste. To identify the compounds responsible for bitterness, we tested all the chemical compounds present in the nutritional supplement for their ability to activate the human TAS2R bitter taste receptors. We found that some compounds are able to activate a small number of TAS2Rs, while others activate several receptors. Our results revealed that it is possible to create new nutritional supplement formulations with reduced bitterness, and therefore lead to a better acceptability.