Gram-positive bacteria protect their cell envelope through d-alanylation of lipoteichoic acid (LTA), a process initiated by DltA-mediated activation of d-alanine and loading onto the carrier protein DltC. Although structural and biochemical studies have established key features of DltA catalysis, direct observation of adenylate formation and carrier protein loading within a reconstituted DltA-DltC system has remained limited. Here, we reconstituted the Bacillus subtilisd-alanylation pathway and used native mass spectrometry to resolve DltA-dependent reaction intermediates and products. We detected ATP-dependent adenylation of d-alanine by DltA followed by transfer to holo-DltC. This process was inhibited by a sulfamoyl-adenosine compound that mimics the adenylate intermediate. Mutational analysis of the conserved DltA P-loop revealed position-specific effects on catalysis, highlighting structural features that govern substrate processing. Together, these findings define regulatory steps in the Dlt pathway and identify opportunities for targeted inhibition. The same strategy should be adaptable to other systems in which transient acyl- or aminoacyl-carrier protein intermediates are difficult to monitor directly.