The adsorption of an antiglutamate dehydrogenase (Anti-GDH) antibody on different surfaces was studied to probe its orientation and bioactivity. Three different situations were investigated: physisorption on a -COOH-terminated thiols self-assembled monolayer (SAM) on gold, covalent grafting on the same SAM using NHS-EDC activation, and physisorption on a -CH3 SAM. The orientation of the antibody was investigated combining time-of-flight secondary ion mass spectrometry and principal component analysis. Several orientations are proposed for each case and compared to the results of biorecognition measurements with the antigen (GDH). At each step, protein layers were characterized ex-situ with polarization-modulated infrared reflection absorption spectroscopy and in situ (i.e., in the liquid phase) with quartz crystal microbalance with dissipation monitoring. Biorecognition measurements showed interesting correlations with proposed protein orientations. The role of hydrophobic and/or electrostatic interactions and that of covalent bonding are discussed to underline the influence of the orientation on the bioactivity of adsorbed Anti-GDH. © 2014 American Chemical Society.