Antibody-based microarrays are a rapidly emerging technology that has advanced from the first proof-of-concept studies to demanding serum protein profiling applications during recent years, displaying great promise within disease proteomics. Miniaturized micro- and nanoarrays can be fabricated with an almost infinite number of antibodies carrying the desired specificities. While consuming only minute amounts of reagents, multiplexed and ultrasensitive assays can be performed targeting high- as well as low-abundance analytes in complex nonfractionated proteomes. The microarray images generated can then be converted into protein expression profiles or protein atlases, revealing a detailed composition of the sample. The technology will provide unique opportunities for fields such as disease diagnostics, biomarker discovery, patient stratification, predicting disease recurrence and drug target discovery. This review describes an update of high-throughput proteomics, using antibody-based microarrays, focusing on key technological advances and novel applications that have emerged over the last 3 years.