Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics.In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors.D escribed here are easily synthesized isotopically labeled desthiobiotin azide (isoDTB) tags that shortened the chemoproteomic workflowa nd allowed an increased coverage of cysteines in bacterial systems.They were used to quantify 59 %o fa ll cysteines in essential proteins in Staphylococcus aureus and enabled the discovery of 88 cysteines that showed high reactivity,w hichc orrelates with functional importance.F urthermore,2 68 cysteines that are engaged by covalent ligands were identified. Inhibition of HMG-CoA synthase was verified and will allow addressing the bacterial mevalonate pathway through an ew target. Overall, ab road map of the bacterial cysteinome was obtained, which will facilitate the development of antibiotics with novel modesof-action.