Background: Diabetes Mellitus (DM) is one of the major health problems and morbidity causes in contemporary society worldwide. Recently, it has been suggested that the addition of citrate in plasma extraction decreases glucose degradation by endogenous enzymes, which could contribute in an important manner to the diagnosis of DM. However, this strategy has not been sufficiently evaluated. The present study aimed to evaluate glucose determination in serum and in plasma obtained with different anticoagulants. Methods: We obtained 20 venous blood paired samples on consecutive days from the same individual, collected in dry tubes with separator gel and in glass tubes with sodium fluoride and EDTA-K₂, either in the presence or absence of sodium citrate. Glucose was determined by an oxidase/peroxidase method using the same system by a unique analyst. Results: Serum determinations were constantly higher than in plasma (P < 0.02), and the addition of citrate increased the analytical variability. Conclusions: Our results were highly relevant statistically, and did not corroborate the addition of citrate in plasma extraction for the inhibition of glycolysis, at least using the standard anticoagulant concentrations.