© 2020 Olson et al. Published by The American Society for Biochemistry and Molecular Biology, Inc. The hexosamine biosynthesis pathway (HBP) branches from glycolysis and forms UDP-GlcNAc, the moiety for O-linked β-GlcNAc (O-GlcNAc) post-translational modifications. An inability to directly measure HBP flux has hindered our understanding of the factors regulating protein O-GlcNAcylation. Our goals in this study were to (i) validate a LC-MS method that assesses HBP flux as UDP-GlcNAc (13C)-molar percent enrichment (MPE) and concentration and (ii) determine whether glucose availability or workload regulate cardiac HBP flux. For (i), we perfused isolated murine working hearts with [U-13C6]glucosamine (1, 10, 50, or 100 μM), which bypasses the rate-limiting HBP enzyme. We observed a concentration-dependent increase in UDP-GlcNAc levels and MPE, with the latter reaching a plateau of 56.3 ∓ 2.9%. For (ii), we perfused isolated working hearts with [U-13C6]glucose (5.5 or 25 mM). Glycolytic efflux doubled with 25 mM [U-13C6]glucose; however, the calculated HBP flux was similar among the glucose concentrations at ∼2.5 nmol/g of heart protein/min, representing ∼0.003-0.006% of glycolysis. Reducing cardiac workload in beating and nonbeating Langendorff perfusions had no effect on the calculated HBP flux at ∼2.3 and 2.5 nmol/g of heart protein/min, respectively. To the best of our knowledge, this is the first direct measurement of glucose flux through the HBP in any organ. We anticipate that these methods will enable foundational analyses of the regulation of HBP flux and proteinO-GlcNAcylation.Our results suggest that in the healthyex vivo perfused heart, HBP flux does not respond to acute changes in glucose availability or cardiac workload.