Applications of peptide arrays in the field of enzyme profiling are described in detail. Focus is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimization for kinases. Additionally, several examples for reliable identification of substrates for other enzymes like lysine methyl transferases and ATP-ribosyltransferases are given. Finally, use of high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological fluids like cell lysates or lysates of complete organisms will be analysed. All published examples of peptide arrays used for enzyme profiling are summarized comprehensively.