Building on a previous method that monitored de novo protein synthesis in mycobacteria using stable-isotope labeling and MALDI-TOF MS, we sought to develop a complementary bottom‑up proteomics workflow capable of deeper molecular resolution. In this follow-up study, we will integrate a metabolic labeling strategy to incorporate NCAAs into NSPs, thereby selectively capturing and analyzing nascent proteomes during antibiotic treatment. This labeling strategy introduces an azide‑functionality, enabling bioorthogonal tagging for selective enrichment. After metabolic incorporation, labeled proteins undergo click‑mediated ligation to biotinylated PEG reporters, and are enriched on neutravidin beads prior to on-bead tryptic digestion. The resulting peptides are analyzed by HPLC‑MS/MS, enabling quantitative profiling of newly synthesized proteins at the peptide level. This bottom-up approach expands the original isotopic MALDI‑TOF MS method by providing deeper coverage, allowing us to resolve how different antibiotics perturb protein synthesis programs in mycobacteria with higher sensitivity and taxonomic precision.