Early mass spectrometry (MS)-based methods to interrogate NRPS pathways originated from related proteomics-based approaches, and therefore they often involve interrogation of the carrier protein or T domain of a pathway. T domains are small in size (< 15 kDa), lending to their facile detection and distinguishable mass shifts when bound to substrates and/or cofactors. There are two types of MS approaches that can be used: top-down or bottom-up. Top-down MS analyzes intact proteins, often stand-alone T domains, though larger constructs have also been successfully detected. Many top-down MS workflows use ion trap instruments with lower resolution or Fourier- transform mass spectrometry (FTMS) with much higher resolution. FTMS is ideal for detecting mass increases as low as 1 Da, but these instruments tend to be custom builds and/or very expensive. In bottom-up MS workflows, in vitro reactions with NRPSs are performed, followed by digestion, either enzymatically with trypsin or chemically with cyanogen bromide. The resulting peptides are separated by reverse phase liquid chromatography (LC) before being individually analyzed by FTMS, often with tandem MS (MS 2 ). Most of these techniques have been discussed elsewhere in more detail, 122-124 but we provide a summary and comparison below. It should be noted that MS in general is a destructive detection method, meaning that samples are measured at an end point and data often cannot be collected in a continuous manner.