Mycobacterium tuberculosis is a professional human pathogen and causative agent of tuberculosis (TB), one of the leading causes of death worldwide. There is currently an incomplete understanding of how M. tb subverts the host immune system and persists within the human body. To bridge these gaps in knowledge, TB researchers are returning to basic research for new perspectives in drug design, vaccine development, and anticipation of drug resistance. N-terminal acetylation (NTA) is an abundant modification found on 70-90% of eukaryotic proteins. This modification has been shown to be biologically relevant in eukaryotic systems, playing roles in protein interactions, stability, localization, and folding. The first virulence factor known to be N-terminally acetylated was the mycobacterial protein, EsxA. EsxA is secreted by the ESAT-6 System-1 (ESX-1) secretion system, which is required for virulence in pathogenic mycobacteria and some Gram-positive pathogens. Importantly, ESX-1 is necessary for virulence in M. tb. ESX-1 is conserved in Mycobacterium marinum, an accepted model for studying certain aspects of virulence in M. tb. Acetylation of EsxA, as well as other ESX-1 substrates, also occurs in M. marinum, suggesting that the mechanism of NTA may be conserved in mycobacteria. However, the biological mechanisms for targeting of NTA on mycobacterial proteins are not known. The work presented here aims to expand the current knowledge of mycobacterial biology and provide insight into the role of NTA in mycobacterial virulence. We employed genetic and proteomic techniques to profile the N-terminal acetylome of M. tb and M. marinum. We show that the mycobacterial genome can be engineered to improve the proteome, allowing mass spectrometry analysis of otherwise intractable regions of a protein (i.e. N-terminus). Next, we developed a novel workflow for the enrichment of mycobacterial protein N-termini. Our approach utilizes a filter assisted sample preparation method compatible with mass spectrometry analysis of mycobacterial protein digests enriched for protein N-termini. We demonstrate that NTA of the mycobacterial proteome is abundant and diverse, which could inform our understanding of how N-terminal acetylation affects mycobacterial metabolism, gene expression and virulence. Using our established N-terminal enrichment approach, we then analyze NTA under conditions that mimic the host environment. Future work in this area will allow us to determine if NTA is a dynamic process and how different carbon sources may alter it. Collectively, this work provides insight into the role of NTA in mycobacteria and advances our understanding of NTA in mycobacterial pathobiology.