Proinsulin is synthesized in the beta-cells of the endocrine pancreas, one of the four cell types found in the islets of Langerhans. Specific enzymatic cleavage of proinsulin results in the formation of equimolar amounts of insulin and C-peptide, via several intermediate split-proinsulin forms. Most mammals produce a single insulin, but in rodents two non-allelic insulin genes are expressed. There is an inverse ratio between the two insulins in rats and mice, the reason for this being unknown. It has been suggested that differences in transcription, translation (biosynthesis) and/or posttranslational processes (enzymatic conversion, intracellular degradation) could be possible explanations. Elevated amounts of proinsulin-immunoreactive material (PIM) have been described to occur in various conditions/diseases, suggesting alterations in beta-cell function, but the composition of the secreted PIM (intact proinsulin or its intermediates) has been incompletely determined. Studies of the biosynthesis of proinsulins and their conversion with the purpose of revealing some of these points depend on accessible reversed-phase high-performance liquid chromatographic (RP-HPLC) analyses capable of separating all the relevant, closely related polypeptides involved. This review will deal with the optimization of the RP-HPLC separations as well as sample preparation and recovery. Applications of the selected methods in the study of proinsulin biosynthesis and its conversion will also be presented.