The altered expression of gangliosides, one of most abundant classes of mammalian glycolipids, in cell plasma membranes coincides with the onset of multiple neuro-degenerative diseases and the metastasis of several cancers, which has spurred interest in these lipids as potential therapeutics and disease biomarkers. Structurally, gangliosides consist of a ceramide lipid tail linked to an oligosaccharide group containing one or more sialic residues. Gangliosides exhibit extensive structural heterogeneity due to variations in length and chemical modification of the lipid moiety and the monosaccharide composition of the oligosaccharide moiety. Both factors have made it challenging to isolate gangliosides from mammalian tissue that possess the structural homogeneity and purity required for many biomedical applications. Conversely, the use of synthetic gangliosides in biomedical research has also been stifled by the complexity of ganglioside synthesis and, in particular, the need to generate the carbohydrate moiety with the correct regio- and stereo-chemistry. The latter often relies on advanced chemistry techniques and tedious purification steps that limit synthetic access to the full range of gangliosides observed in nature. Thus, biomedical applications of gangliosides stand to benefit from a cell-free synthetic tool that facilitates assembly of the glycan headgroup onto the lipid core. To realize the on-demand assembly of ganglioside glycans directly on a lipid anchor, I developed a biomimetic synthesis platform modeled after the Golgi apparatus, the natural site of glycolipid biosynthesis in eukaryotes. The Golgi apparatus exhibits exquisite control over glycan synthesis owing to its arsenal of glycosyltransferase (GT) enzymes and their localization within sub-Golgi membrane compartments. This project sought to recapitulate these three critical features of the Golgi – glycosyltransferases, the membrane environment, and enzyme compartmentalization – on-chip to provide a cell-free, Golgi-like assembly line (aka ‘Minimal Golgi’) for spatiotemporally controlled glycan synthesis. For this dissertation, I developed a prototype of the Minimal Golgi platform that integrates recombinant GTs, membrane mimics (e.g. supported lipid bilayers), and modular reaction units to perform multi-step glycosylation of lipid substrates. As a proof-of-concept, I used the prototype device to carry out the two-step conversion of ganglioside GM3 to ganglioside GM1 using recombinant bacterial GTs. In Chapter 1, I introduce the biology of glycolipid biosynthesis in the Golgi and describe tools that may be used to mimic essential biophysical and biochemical features of this process in vitro. In Chapters 2 and 3, I provide a comprehensive review of the current state-of-the-art technologies for glycan synthesis and analysis on-chip. In Chapter 4, I detail the development of the Minimal Golgi chip and demonstrate the serial enzymatic glycosylation of lipids embedded in unilamellar vesicles (i.e., liposomes) and planar-supported lipid bilayers. With this system, I demonstrate a versatile platform that can be used to (a) mimic and investigate regulatory mechanisms of the native Golgi and (b) biosynthesize lipid-linked glycans within a spatiotemporally-controlled environment. In Chapter 5, I summarize the major outcomes of the work described in Chapters 2 – 4 and propose future developments and applications of the Minimal Golgi platform.