The ability to measure the progress of a reaction over time is necessary in general biochemistry. Some reactions consist of a single step and generate few if any intermediates, but the adenylation reaction is a two-step process with multiple reactants, products, and cofactors. This makes measurement of adenylation activity possible at many points along the reaction pathway (Figure ), but it is important to understand each minor event and how different assays account for the dynamic interactions between multiple substrates, cofactors and other enzymes over the course of the reaction. Specifically, adenylation begins when an A domain binds both ATP and its cognate substrate in adjacent active site pockets. Hydrolysis of ATP produces PPi, which exits the active site, and an AMP-tethered substrate. The adenylated substrate remains in the active site until a nucleophilic attack catalyzed by the Ppant arm of a neighboring T domain, at which time the high-energy substrate-AMP is transferred to the free thiol group of Ppant, releasing AMP from the active site. There are many proposed conformational rearrangements of A domains and T domains that must take place for each step of the reaction cycle to proceed, and many of these conformations have been captured in the structural studies discussed above. The order of various techniques presented in the following section begins with direct methods of substrate detection and ends with indirect methods, moving in reaction chronology (Figure ). Some strategies that will not be discussed in detail here include the body of work pioneered by the Ishikawa and Kakeya labs in the realm of chemical proteomics and activity-based protein profiling. Building on work that designed and synthesized clickable probes and inhibitors of A domains, Ishikawa and coworkers have established methods that can detect and interrogate endogenous NRPSs using ELISA and in vivo techniques. This has provided valuable insight into chemical moieties required for binding and remodeling of active site architecture, while avoiding the labor-intensive and problematic enzyme purifications that many other workflows require. These contributions, while significant to the field, are often untargeted or exploratory in nature and require the generation of synthetic probes for each tested substrate. Thus, the workflow is not amenable to the comprehensive study of substrate selectivity in previously identified A domains of interest. The methods we have chosen to focus on are broadly applicable to any adenylation enzyme activating any acyl-containing substrate and can be performed without extensive technical knowledge. Many of the assays discussed in this section have been summarized elsewhere, but to the best of our knowledge this is the most complete review of biochemical methods for analysis of the adenylation reaction.