To determine cAMP, cGMP, or cyclic nucleotide analog levels, this chapter develops a method to first separate the analog(s) from cAMP or cGMP by a simple chromatographic procedure followed by functionally directed assay of the respective cyclic nucleotide by either cAMP- or cGMP-dependent protein kinase activation. The sensitivity is much better than that of the cyclic nucleotide binding assays, and approaches that of the radioimmunoassays. Because high concentrations of cyclic nucleotide analogs are usually used for intact tissue incubations, trace cyclic nucleotide contaminants in commercial cyclic nucleotides may not be separated during purification and would thus interfere with the assay for the nucleotide of interest. It is therefore necessary to purify the analogs before use. Alternatively, sensitive and specific cAMP- and cGMP-dependent protein kinase activation assays are described in the chapter.