Crabtree-positive yeasts rapidly consume glucose via glycolysis, making it difficult to experimentally estimate their actual glycolytic rate or flux. We present a stable isotope labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protocol to quantitatively estimate glycolytic and related carbon metabolic fluxes using Saccharomyces cerevisiae. This approach defines time windows to capture glucose metabolic intermediate production before label saturation, enabling a comparison of glycolytic flux changes across different cells. This protocol provides a reliable, quantitative approach to study dynamic metabolic fluxes in these cells. For complete details on the use and execution of this protocol, please refer to Vengayil et al., 2024..