This review summarizes the major activities in the field of protein microarray technology. A short summary of the theoretical concepts of miniaturized ligand binding assays explains why such microspot assays represent the most sensitive approaches for capture-target assays. The main focus of this review is centered on the applications using miniaturized and parallelized protein binding assays which rely on the product formation between immobilized capture molecules and their corresponding target molecules which are present in the sample. These types of ligand binding assays are useful tools for protein identification, quantification and protein affinity studies. Protein identification and quantification assays have a great potential in the field of diagnostics and proteomics where many different protein markers which are present in complex samples have to be analyzed in parallel. Protein affinity assays can be used to analyze interactions between proteins such as antibodies, receptors or enzymes with other proteins, peptides, low molecular weight compounds, oligosaccharides or DNA. Different applications of protein microarray-based assays and their huge potential for diagnostic and proteomic approaches will be discussed.