Building on a previous method that monitored de novo protein synthesis in mycobacteria through 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 BONCAT and THRONCAT, two metabolic labeling strategies that incorporate non‑canonical amino acids into newly synthesized proteins (NSPs) to selectively capture and analyze nascent proteomes during antibiotic treatment. BONCAT introduces azide‑functionalized methionine analogs, whereas THRONCAT employs alkyne‑bearing threonine analogs, 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 peptide-level resolution. This bottom-up approach expands the original isotopic MALDI‑TOF method by providing deeper coverage and improved specificity, allowing us to resolve how different antibiotics perturb protein synthesis programs in mycobacteria with higher sensitivity and taxonomic precision.