Hydrogen peroxide (H2O2) is an important regulator of cellular events leading to glucose transport activation in mammalian skeletal muscle. In the absence of insulin, H2O2 in the low micromolar range engages the canonical IRS-1/PI3K/Akt-dependent insulin signaling pathway, as well as other signaling elements (AMPK and p38 MAPK), to increase basal glucose transport activity. In contrast, in the presence of insulin, H2O2 antagonizes insulin signaling by recruitment of various deleterious serine/threonine kinases, producing a state of insulin resistance. Here, we describe the H2O2 enzymatic-generating system, utilizing glucose oxidase, that has been used to investigate the impact of H2O2 on cellular signaling mechanisms related to glucose transport activity in isolated rat skeletal muscle preparations, such as the soleus. By varying the glucose oxidase concentration in the medium, target ranges of steady-state H2O2 concentrations (30-90 μM) can be attained for up to 6 h, with subsequent assessment of cellular signaling and glucose transport activity.