Antibody microarrays have the potential to revolutionize protein diagnostics. The major problems in the fabrication of antibody arrays, however, concern the reproducibility and homogeneity of the attachment of the proteins on the solid substrate. We here compare the DNA-directed immobilization (DDI) method with two conventional strategies for immobilization of antibodies on glass substrates. DDI is based on the self-assembly of semisynthetic DNA-streptavidin conjugates which converts an array of DNA oligomers into an antibody microarray. DDI was compared with direct spotting of antibodies on chemically activated glass slides and with immobilization of biotinylated antibodies on streptavidin-coated slides. The immobilized antibodies were used as capture reagents in a two-sided (sandwich) immunoassay for the quantification of rabbit IgG as a model antigen. Detection limits down to 0.001nM (150 pg/mL) were attained with all three array formats; however, DDI and direct spotting of the antibodies led to the highest fluorescence intensities. DDI led to the best spot homogeneity and intra- and interexperimental reproducibility. Moreover, DDI allowed highly economical use of antibody materials; that is, at least 100-fold less antibody is needed for preparing an array by DDI instead of by direct spotting. Taking into account the greater versatility and convenience of handling of the self-assembly approach, this study demonstrates that DDI is an advantageous alternative for generating versatile and robust protein arrays.