Technical Abstract: Measurements of nonstructural carbohydrates (NSC) in plant tissues are important to estimate plant organ resources available for plant growth and stress tolerance or for feed value to grazing animals. A popular commercially available assay kit used to detect glucose with a light sensitive dye reaction was recently discontinued and replaced by a test-tube scale kit that assays glucose through enzymatic coupled reactions and the formation of reduced nicotinamide adenine dinucleotide (NADH). A laboratory microplate enzymatic method was developed for the new glucose kit and evaluated for rapidly assaying NSC components, including glucose, fructose, sucrose, fructans and starch, in cool-season perennial grasses. By standard addition, dilution, and temporal tests of enzyme reactions, we found that this microplate enzymatic assay is a rapid and reliable method to quantify NSC composition in grass forage samples. The microplate method allows analysis of many samples per day and considerably improved time and reagent use efficiencies, especially for a large number of samples. In addition to forage, this method should be suitable for measuring NSC concentrations in fresh or dry tissues of a variety of other plant samples.