Several proteins of viral and cellular origins are myristoylated on an amino-terminal glycine residue during biosynthesis. The enzyme responsible for this modification, myristoyl CoA:protein N-myristoyltransferase (NMT), can be measured in cell-free systems by following the transfer of [3H]myristate from [3H]myristoyl CoA to a synthetic peptide substrate. We report here a procedure for the analysis of NMT activity using ion-exchange chromatography on CM-Sepharose to separate [3H]myristoyl peptide from radiolabeled reactants. This technique provides a convenient method for assaying multiple samples that is much more rapid and sensitive than procedures that rely on reversed-phase HPLC for the separation of reaction components. Characterization of this assay indicates that it is suitable for the kinetic analysis of NMT activity and for the rapid analysis of column fractions generated during the purification of NMT.