Pseudomonas aeruginosa is an opportunistic pathogen, which causes significant and difficult to eradicate infections that can become highly antimicrobial resistant. Understanding the cellular mechanisms behind the high-level resistance is critical for the future production of novel antimicrobial treatments. Peptidoglycan (PG) is an integral component of bacterial cell walls that participates in many cellular functions, is essential for survival, and is a common target for antimicrobials. Aspects of PG structure are fairly conserved across all bacteria. However, there is also considerable variation and numerous modifications in PG that can occur between species and in response to environmental stimuli. Examining a global overview of PG composition would help us understand this complex biopolymer and potentially identify unique mechanisms of antimicrobial resistance. However, conventional methods for analyzing PG composition are difficult and time-consuming. For this thesis, I applied modern bioinformatic techniques to develop a peptidoglycomics workflow and tested the sensitivity by examining two distinct growth morphologies, biofilm and planktonic, that differ in the overall resistance to antimicrobials. Then I used this methodology to examine the PG composition of the P. aeruginosa epidemic strains which are highly virulent with enhanced antimicrobial resistance. In addition, I examined the effect of nutritional conditions on PG composition. Overall, I demonstrate that my peptidoglycomics workflow produced a highly sensitive and detailed analysis of PG composition and I identified several modifications that have not been previously identified in P. aeruginosa. Further, my analyses show that P. aeruginosa can vary the composition of the PG significantly depending on the strain, nutrient condition, and growth morphology. Some of these changes may have implications for the virulence and antimicrobial resistance of P. aeruginosa. In addition, I provide an initial characterization of a PG modification that is likely important for the biofilm growth morphology. Together, this study has demonstrated the sensitivity of this peptidoglycomics methodology and has significantly advanced our understanding of the PG in P. aeruginosa. Future use of this methodology will facilitate further understanding of the dynamics of PG production and modification, as well as how it contributes to overall cellular functions.