Uniformly deuterated sterols and biosynthetically related materials are important for neutron, NMR, tracing and bioanalysis studies as well as critical tools for the creation of improved lipid nanoparticle formulations. The production of sufficient quantities of materials relies not only on the engineering of microorganisms to selectively accumulate desired materials but also methods for the isolation, purification and characterisation of these materials to ensure their usefulness. Uniformly deuterated squalene, the universal precursor to sterols in biological systems, has been produced and characterised. Cholesterol has been produced with controlled levels of uniform deuteration, increased biosynthetic yield and a methodology developed for the extraction and purification of this material without HPLC. Two sterols, not previously produced in deuterated forms, have been prepared with uniform deuteration: 22,23-dihydrobrassicasterol and 24-methylenecholesterol. This report triples the number of sterols that have been produced with uniform deuteration, purified and characterised and provides a silylation/silver ion chromatography protocol for the separation of sterols which differ by the degree of unsaturation. The techniques for the 13C NMR analysis of deuterated sterols, site-specific deuteration levels and an analysis of key biosynthetic steps based on these data are reported.