Actinoplanes teichomyceticus produces teicoplanin (Tcp), a "last resort" lipoglycopeptide antibiotic used to treat severe multidrug resistant infections such as methicillin-resistant Staphylococcus aureus (MRSA). A number of studies have addressed various steps of Tcp biosynthesis using in vitro assays, although the exact sequence of Tcp peptide core tailoring reactions remained speculative. Here, we describe the generation and analysis of a set of A. teichomyceticus mutant strains that have been used to elucidate the sequence of reactions from the Tcp aglycone to mature Tcp. By combining these results with previously published data, we propose an updated order of post-assembly line tailoring processes in Tcp biosynthesis. We also demonstrate that the acyl-CoA-synthetase Tei13* and the type II thioesterase Tei30* are dispensable for Tcp production. Five Tcp derivatives featuring hitherto undescribed combinations of glycosylation and acylation patterns are described. The generation of strains that produce novel Tcp analogues now provides a platform for the production of additional Tcp-like molecules via combinatorial biosynthesis or chemical derivatization.