Background: Antibody microarrays (Ab-array) represent a new, innovative proteomics platform for high-throughput protein expression profiling in body fluids. Because they allow for multiplexed measurements in small sample volumes, Ab-arrays are an interesting alternative to conventional indirect sandwich immunoassay (ELISA or DELFIA) tests in clinical or population screening if sets of markers are to be analyzed simultaneously. However, to allow implementation of Ab-arrays in clinical or population screening programs, it is of vital importance to establish that this method is both sensitive and quantitative. Methods: This study developed and optimized a duplex Ab-array for pregnancy-associated plasma protein A (PAPP-A) and human chorion gonadotropin (fβ-hCG), two serum biomarkers currently analyzed by conventional biochemical techniques in prenatal screening. Serum samples from pregnant women, representing the dynamic range of both markers, were analyzed on Ab-arrays, and validated to the, in prenatal screening routinely applied, AutoDelfia system. Results: Two different array hybridization conditions were tested, i.e., direct and indirect labeling, of which the indirect method displayed a sensitive and quantitative performance and a low intra- and inter-assay variation. Conclusions: Taken together, these findings indicate that Ab-array technology is a promising alternative for ELISA or DELFIA in population screening programs, allowing future quantitative analysis of multiple biomarkers simultaneously in small volumes of serum.