Corynetoxin complex (CT) is a family of toxins responsible for Annual Ryegrass Toxicity (ARGT) and is closely related in structure to tunicamycin (TM) antibiotics. This paper describes high-performance liquid chromatography (HPLC) methods (reversed-phase and normal-phase) that give improved resolution of individual CT components. The normal-phase method separates CT into H series components and a mixture of S and U series components, while the reversed-phase system, after removal of H series toxins, can resolve the n, i and a isomers of the S and U series. Catalytic reduction provides further evidence of the structures of S and U series members. These HPLC systems are also applicable to other tunicaminyluracil-based antibiotics like TM (with acids ranging from C14 to C17) and uracil-containing streptovirudin (SV, C12 to C15), allowing separation and tentative assignment of previously unrecognised components (including C10, C11 and C16 SVs). The described HPLC systems provide an improved means of analysing tunicaminyluracil antibiotics and a way to assess samples for the presence of H-series components using normal-phase chromatography.