Congenital disorders of glycosylation (CDGs) are rare inherited disorders affecting glycosylation of proteins and lipids and sharing very heterogeneous multivisceral symptoms. The biochemical screening of these diseases is currently limited to electrophoresis or HPLC separation/quantification of serum transferrin glycoforms and is relatively frequently hampered by genetic polymorphism. Further, it has been shown that transferrin glycosylation can be very poorly affected in confirmed CDGs. We developed a fast and simple two-dimensional (2-DE) Western-blot analysis applied to the simultaneous detection of various serum glycoproteins, i.e. haptoglobin, α1-anti-trypsin, transferrin and α1-acid glycoprotein, and applied it to a large cohort of CDGs and secondary glycosylation disorders. When separated using 2-DE, haptoglobin β glycoforms showed clear abnormalities in all interpretable CDG type I and CDG type II patterns. Although secondary glycosylation defects such as alcoholism, untreated fructosemia and bacterial neuraminidase remain to be excluded, we showed that 2-DE pattern of haptoglobin β glycoforms thus constitute a very reliable additional biomarker of all types of CDGs. Coupled with common screening techniques and glycans mass spectrometry, it can orientate and facilitate the way towards CDG molecular diagnostic.