The human proteome is known to contain >500 protein kinases, which regulate almost all facets of cellular biology by the post-translational attachment of a phosphate moiety to serine, threonine, or tyrosine residues within a substrate protein. Most protein kinases remain poorly characterized and, as a result, current studies are directed toward defining their target substrates experimentally to gain a comprehensive view of the signaling proteins and pathways modulated by these kinases. Herein, we describe a rapid and convenient method for elucidating the consensus substrate motif for phosphorylation by a protein kinase using peptide SPOT arrays that are custom-synthesized on a cellulose membrane support. The definition of the target consensus motif provides an important starting point for the identification of physiologically relevant kinase substrates.