Congenital disorders of glycosylation (CDG) are inherited diseases that can affect not only the N‐glycan (e.g. CDG type I and II) but also the O‐glycan biosynthesis pathway. In the absence of specific clinical symptoms, there is a need for a reliable biological screening of these two groups of CDG. Using a few microlitres of human serum, 2‐DE and immunoblotting were applied to the separation and simultaneous detection of the isoforms of the O‐glycosylated protein apolipoprotein C‐III (apoC‐III) and of four N‐glycosylated proteins, namely alpha‐antitrypsin, alpha‐1 acid glycoprotein, haptoglobin and transferrin. For the study of O‐glycosylation, this technique allowed the reliable separation of the three fractions of apoC‐III and the determination of normal percentage values in an adult population. Concerning N‐glycosylation, the study of serum samples from patients with CDG type Ia revealed marked abnormalities systematically affecting the four 2‐DE separated N‐linked glycoproteins. 2‐DE coupled to immunoblotting using a mixture of specific antibodies could be easily and reliably employed for the combined screening of both N‐ and O‐glycosylation disorders in humans.