d-Cycloserine (DCS) is only used with multidrug-resistant strains of tuberculosis because of serious side effects. DCS is known to inhibit cell wall biosynthesis, but the in vivo lethal target is still unknown. We have applied NMR-based metabolomics combined with principal component analysis to monitor the in vivo effect of DCS on Mycobacterium smegmatis. Our analysis suggests DCS functions by inhibiting multiple protein targets.