In this study we have performed a comparison of the most used spectroscopic glucose-oxidase assay for routine clinical monitoring of serum glucose, with the high-performance anionexchange chromatographic method using pulsed amperometric detection (HPAEC-PAD). The linearity, repeatability and internal reproducibility of the chromatographic method were determined. The correlation by linear regression and difference plot was analyzed. It is known that the enzymatic method leads to unreliable results when glucose must be determined in samples containing high concentration of bilirubin or lipids. On the contrary, the accuracy of the HPAEC-PAD method with these complex matrix sera resulted similar to normal samples showing no interferences. Another advantage was the minimal amount of human serum needed: one microliter was largely sufficient to perform the whole analysis. This fact would make the HPAEC-PAD method applicable when samples of preterm infants or new born babies of low weight must be analyzed.