Breast cancer (BC) remains one of the leading causes of mortality among women, with triple-negative breast cancer (TNBC) standing out for its aggressive nature and limited treatment options. Metabolic reprogramming, one of cancer’s hallmarks, underscores the importance of targeting metabolic vulnerabilities for therapeutic intervention. This study aimed to investigate the impact of de novo serine biosynthetic pathway (SSP) inhibition, specifically targeting phosphoglycerate dehydrogenase (PHGDH) with NCT-503, on three TNBC cell lines: MDA-MB-231, MDA-MB-468 and Hs 578T. First, MS-based proteomics was used to confirm the distinct expression of PHGDH and other SSP enzymes using the intracellular proteome profiles of untreated cells. Furthermore, to characterize the response of the TNBC cell lines to the inhibitor, both in vitro assays and label-free, bottom-up proteomics were employed. NCT-503 exhibited significant cytotoxic effects on all three cell lines, with MDA-MB-468 being the most susceptible (IC50 20.2 ± 2.8 µM), while MDA-MB-231 and Hs 578T showed higher, comparable IC50s. Notably, differentially expressed proteins (DEPs) induced by NCT-503 treatment were mostly cell line-specific, both in terms of the intracellular and secreted proteins. Through overrepresentation and Reactome GSEA analysis, modifications of the intracellular proteins associated with cell cycle pathways were observed in the MDA-MBs following treatment. Distinctive dysregulation of signaling pathways were seen in all TNBC cell lines, while modifications of proteins associated with the extracellular matrix organization characterizing both MDA-MB-231 and Hs 578T cell lines were highlighted through the treatment-induced modifications of the secreted proteins. Lastly, an analysis was conducted on the DEPs that exhibited greater abundance in the NCT-503 treatment groups to evaluate the potential chemo-sensitizing properties of NCT-503 and the druggability of these promising targets.