The emergence of multidrug-resistant (MDR) bacteria has become one of the greatest threats to global public health and infectious diseases rank among the leading causes of mortality worldwide. Without urgent action, the rates of death caused by incurable infections every year are expected to rise more than tenfold by 2050, reaching 10 million every year, surpassing deaths caused by cancer. This situation is aggravated by the diminishing antibiotic pipeline as pharma has perceived this area to be uneconomical and as such, there are now fewer than 50 antibiotic compounds in development, while the cancer drug pipeline currently has more that 800 candidates in the pipeline. This highlights the need to develop and discover new antibiotics with novel mechanisms and new chemical scaffolds. Daptomycin is a cyclic lipodepsipeptide natural product, exhibiting potent antimicrobial activity against highly resistant Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and several species of Streptococci. It is a non-ribosomally synthesised peptide consisting of thirteen amino acids, six of which are non-proteinogenic residues: (S)-2-Amino-4-(2-aminophenyl)- 4-oxo-butanoic acid (Kynurenine, Kyn13), (2S,3R) MeGlu12, D-Ser11, D-Ala8, Orn6 and D-Asn2. Ten of the thirteen amino acids form a 31-membered depsipeptide ring, with an ester bond between the side-chain of Thr4 and the carboxylic acid of Kyn13. The remaining exocyclic tripeptide is then capped at the N-terminus with an octanoyl fatty acid. Previous chemoenzymatic synthesis, semi-synthesis, and combinatorial biosynthesis only produced a very limited number of daptomycin analogues, leaving much of the chemical space unexplored. We describe a novel synthesis of native daptomycin using Boc-based SPPS and employing a key on-resin ozonolysis step to install the difficult Kyn residue. The methodology was extended to prepare several novel daptomycin analogues that were evaluated for activity against the Gram positive pathogen, Staphylococcus aureus.