Daptomycin is the first cyclic lipopeptide antibiotic approved by the FDA in 2003 for treating skin and skin structure infection and right-side endocarditis. 1 Its activity against multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) makes daptomycin a good target for derivatization to generate next-generation antibiotics to combat the foreseeable emergence of superbugs that all the current available antibiotics fail to kill. The first total synthesis of daptomycin was established by our group in 2013 utilizing Serine/Threonine Ligation (STL).2 Since then, we have been making more than 100 analogues of daptomycin with modifications at various sites which most of them can only be achieved by total synthesis. From the library of daptomycin analogues we prepared, we established a comprehensive structure-activity relationship (SAR) of daptomycin . Based on the SAR, we designed and synthesized more daptomycin analogues and identify several analogues with improved efficacy. Particularly, HY2371 showed superior activities against Enterococci tested both in in vitro and in vivo studies compared to daptomycin.