Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals. Due to their critical role in cellular growth and metabolism, GLUT dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUT have proven to be difficult to implement, hampering deeper mechanistic studies. Indeed, our understanding of the role of GLUT transporters to whole-body glucose homeostasis is largely derived from the kinetics of 2-deoxy-glucose and not D-glucose, as this sugar is rapidly phosphorylated in the cell. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5, and demonstrate how inaccuracies can be generated when assessing the impact of mutations and inhibitors with suboptimal activity. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids with a small number of synthetic lipids. Subsequently, we show how GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar KM, but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high KM for D-glucose and yet a very slow turnover, which might be to ensure D-glucose uptake is regulated by insulin-dependent trafficking. Overall, our study provides a much-needed transport assay for analyzing in vitro GLUT activities, and implies that high-levels of free fatty acids in membranes, as found in those suffering from metabolic disorders, are a likely contributor to the impairment of glucose transport.