Daptomycin (DAP) is a calcium (Ca2+)‐dependent FDA‐approved antibiotic drug for the treatment of Gram‐positive infections. It possesses a complex pharmacophore hampering derivatization and/or synthesis of analogues. To mimic the Ca2+‐binding effect, we used a chemoenzymatic approach to modify the tryptophan (Trp) residue of DAP and synthesize kinetically characterized and structurally elucidated regiospecific Trp‐modified DAP analogues. We demonstrated that the modified DAPs are several times more active than the parent molecule against antibiotic‐susceptible and antibiotic‐resistant Gram‐positive bacteria. Strikingly, and in contrast to the parent molecule, the DAP derivatives do not rely on calcium or any additional elements for activity. Ca not required: Daptomycin relies on calcium as an essential element for its strong antibiotic activity. Herein, a chemoenzymatic approach is used to synthesize regiospecific daptomycin derivatives that are independent of calcium and are more active than the parent drug against daptomycin‐susceptible and daptomycin‐resistant Gram‐positive bacteria.