Glycoprotein modifications in the glycan moiety can occur in diseases such as cancers, inflammatory processes and alcoholism. We combined high‐resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) with lectin affinoblotting in order to establish the normal human plasma glycoprotein map. Human plasma proteins were separated by mini 2‐D PAGE (7 × 9 cm), transferred onto polyvinylidene difluoride membranes and incubated with biotinylated lectins. We focused our study on lectins binding sialic acid and galactose residues. Known plasma glycoproteins such as αl‐antitrypsin, αl‐antichymotrypsin, α2‐HS glycoprotein, αl‐acid glycoprotein, haptoglobin β‐chain and transferrin were easily detected in ng amounts. This protocol was adequate to establish a normal plasma glycoprotein map and will allow the study of glycoproteins in diseases.