Understanding protein-protein interactions is a key step in unravelling the roles proteins play in cellular function. The ability to analyse protein-protein interactions rapidly and economically is a powerful research tool. Using peptide SPOT arrays, peptides of known sequence can be synthesized directly in discrete spots on a cellulose membrane and assayed for an interaction with a protein of interest. Several hundred peptides can be synthesized on each cellulose membrane; therefore, this method is amenable to designing high-throughput peptide binding studies. SPOT arrays are particularly well suited for deducing peptidic binding motifs within proteins that are difficult to purify in sufficient quantities for traditional biochemical analyses, as well as for determining binding specificities and targets for proteins of undefined function. Peptide SPOT arrays have been used extensively to define protein-protein interaction surfaces. In this chapter, we will outline the steps involved in designing and probing a peptide SPOT array to identify peptide binding motifs for a protein of interest.