In the present work, a simple automated microplate method based on cupric ion reduction is described for determining total reducing sugars in food extracts and synthetic serum. The reaction of Cu(II)-Nc (cupric-neocuproine) with reducing sugars was performed in alkaline medium in microplates, and the absorbance of the formed highly colored Cu(I)-Nc chelate in a plate reader at 450 nm was recorded. The proposed method was applied to reducing sugars (glucose, fructose, galactose, maltose, and lactose) and their linear calibration curves were constructed. The detection and quantification limits (LOD and LOQ) for glucose were 0.14 and 0.46 mu M, respectively. Absorbances of glucose were linear within the concentration range 2.5-54.2 mu M and the method showed high linearity (r = 0.9998) over a relatively broad concentration range of analyte. This automated microplate method was validated through linearity, additivity, precision (RSD%, 2.33-6.65), and recovery (101%-103%), revealing that the method is reliable and robust for determining reducing sugars. Total reducing sugar contents of synthetic sugar mixtures, fruit juices, milk, and synthetic serum samples were successfully determined with the proposed method. The results were compared to those of the conventional alkaline Cu(II)-Nc spectrophotometric method. The proposed method offers many advantages when compared to classical methods, such as (sample and reagent) volume reduction (20-fold), simplicity, multiple sample analysis (32 samples in 4 h), and environmental friendliness.