The GE81112 series, consisting of three naturally occurring tetrapeptides and synthetic derivatives, is evaluated as a potential lead structure for the development of a new antibacterial drug. Although the first total synthesis of GE81112A reported by our group provided sufficient amounts of material for an initial in depth biological profiling of the compound, improvements of the routes toward the key building blocks were needed for further upscaling and structure–activity relationship studies. The major challenges identified were poor stereoselectivity in the synthesis of the C-terminal β-hydroxy histidine intermediate and a concise access to all four isomers of the 3-hydroxy pipecolic acid. Herein, we report a second-generation synthesis of GE81112A, which is also applicable to access further representatives of this series. Based on Lajoie’s ortho-ester-protected serine aldehydes as key building blocks, the described route provides both a satisfactory improvement in stereoselectivity of the β-hydroxy histidine intermediate synthesis and a stereoselective approach toward both orthogonally protected cis and trans-3-hydroxy pipecolic acid.