For the uninterrupted observation of natural product bioassembly on nonribosomal peptide synthetases, Quadrupole Fourier Transform Mass Spectrometry (Q-FTMS) was utilized to directly interrogate peptides harboring covalently modified residues in yersiniabactin synthetase. After proteolysis in CNBr, the peptides corresponding to each carrier site were identified and visualized using a continuous kinetic assay. Overall, complex intermediate formation was rapid, with observation of the HPTT-beta-keto-2,2-dimethyl-S-ACP intermediate within 4 s, while each active site reached saturation within approximately 20 s. Reduction of the beta-keto group at the ACP domain was found to have the slowest rate, accumulating only after 40 s. This represents the first study to correlate five active sites in tandem with kinetic and structural resolution of the complex intermediates in addition to regiospecific information preserved in the assay.