Antibody arrays hold great promise for biomedical applications, but they are typically manufactured using chemically functionalized surfaces that still require optimization. Here, we describe novel hetero-functionally activated glass surfaces favouring oriented antibody binding for improved performance in protein microarray applications. Antibody arrays manufactured in our facility using the functionalization chemistries described here proved to be reproducible, stable and also showed good signal intensities. As a proof-of-principle of the glass surface functionalization protocols described in this report, we built antibody-based arrays functionalized with different chemistries that enabled the simultaneous detection of seventy one human leukocyte membrane differentiation antigens commonly found in peripheral blood mononuclear cells. Such detection is specific, semi-quantitative and can be performed in a single assay under native conditions. In summary, the protocol described here, based on the use of antibody array technology enabled the concurrent detection of a set of membrane proteins under native conditions in a specific, selective and semi-quantitative manner and in a single assay.