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The detection and characterization of the phosphatases responsible for the dephosphorylation of N-phosphoryl groups, such as phospholysine, in proteins has been frustrated in large measure by a lack of suitable substrates for the assay of these enzymes. Herein we describe the preparation of phospholysine-containing amino acid homo- and heteropolymers by chemical means, and their use in a simple, nonradioactive assay for phospholysine phosphatase activity