Finally, two in vitro methods have been developed for the quantification of AMP released from an A domain in the second half-reaction. A continuous colorimetric AMP release assay is achieved by coupling the adenylation reaction to NADH consumption. The decrease in NADH is measured with the addition of adenosine kinase (ADK), pyruvate kinase (PK) and lactate dehydrogenase (LDH). Unfortunately, this assay suffers from similar issues as other NADHcoupled assays, namely high background activity and reliance on three additional enzymes. To avoid these issues, a novel discontinuous AMP release assay was developed by Staeben et al. that uses immunodetection of AMP and far red fluorescence polarization for quantification. This assay, though not widely used in the NRPS community, displays good sensitivity and avoids having to couple adenylation activity to one or more additional enzymes. The assay reagents were shown to be stable for 24 hours at room temperature, but the cost and specific handling of antibodies, as well as the instrumentation required, make this protocol somewhat less accessible.