The role of oxidative stress in the pathogenesis of type 2 diabetes (T2D), especially pancreatic β-cell dysfunction and death, has become apparent in the last two decades. Peroxidase- and catalase-based antioxidant mechanisms are particularly weak in β-cells and can be easily overwhelmed by excessive production of reactive oxygen and nitrogen species in the course of pathological processes. Recent research has attempted to define in detail the mechanistic aspects of oxidative stress-induced β-cell dysfunction. Here, we describe the procedures for the measurement of various parameters important to assess oxidative stress in pancreatic β-cells. Detailed protocols for determination of nitric oxide (NO) production, the glutathione redox status, and general oxidative status in β-cells are presented in this chapter.