The elucidation of the manner in which proteins interact with other proteins or nucleic acids can lead to new drug and therapeutic leads in biological systems like signal transduction, protein targeting, and cell cycle control. Moreover, it aids in the design of experiments like mutagenesis and competition for binding sites and computer models to shed light on multi-protein interaction networks. Protein arrays are instrumental to analyze the interactions within complex protein networks in order to address these key questions in cellular biology. In this review, we summarize recent achievements in the field of solid-supported peptide arrays generated by the SPOT method, which allows for fast parallel manual or automated Fmoc-synthesis of arrays of overlapping oligopeptides on cellulose membranes. These arrays represent a powerful tool for the investigation/mapping of antibody epitopes, protein-protein interaction sites, protein-DNA interactions, and most recently protein-RNA interactions. We explain the method, provide an overview over the applications of the technique, and present recent experimental data, like the identification of a peptide sequence, that interferes with packaging of HIV-1 genomes and virus replication. Moreover, we summarize recent developments in the array formats used and discuss future perspectives of the method.