The AMP-Glo Assay is a homogeneous assay that generates a luminescent signal from any biochemical reaction that produces AMP as a reaction product. This versatile system can measure the activity of a broad range of enzymes, such as cyclic AMP-specific phosphodiesterases, aminoacyl-tRNA synthetases, DNA ligases and ubiquitin ligases or enzymes modulated by AMP. The AMP-Glo Assay is designed to quantitatively monitor the concentration of AMP in a biochemical reaction in a wide range of plate formats, including high-throughput formats. The stable luminescent signal of the assay eliminates the need for an injector-equipped luminometer and allows batch-mode processing of multiple plates. The assay can be used to determine the AMP produced either in the presence or absence of ATP as a substrate. The assay contains two reagents: one to terminate the AMP-generating enzymatic reaction and simultaneously remove ATP and convert AMP produced into ADP, and a second reagent that converts the ADP to ATP followed by conversion of the ATP into a luminescent signal using the luciferin/luciferase reaction. The assay also is well suited for monitoring AMP produced in biochemical reactions catalyzed by enzymes that do not use ATP as a substrate, such as cAMP-dependent phosphodiesterases (PDE) and bacterial DNA ligases. The AMP-Glo Assay has a high dynamic range and produces a strong signal at low substrate conversion, making it well suited for screening low activity enzymes. The assay produces minimal false hits and Z´ values greater than 0.7.