The challenges involved in the chemical synthesis of insulin and its analogs hinder the exploration of the structure-function relationships of this indispensable therapeutic. Problematic features such as the hydrophobicity of the A-chain and the complexity of the disulfide architecture are unique to insulin and have required implementation of specialized strategies. Our efforts have centered on two fundamental approaches: the single chain biomimetic route and the two-chain directed disulfide method. An example of the former is our use a novel single chain precursor “desDi” which enabled superior folding efficiency and proved to be less sensitive to individual substitutions. The latter approach is exemplified by the directed disulfide approach using orthogonally protected cysteine pairs to form insulin. We have additionally expanded the scope of the two-chain directed disulfide method to other insulin like peptides which contain oxidation-sensitive residues through the use of an iodine-free method employing Phacm cysteine protection and enzymatic conversion to form the (A7-B7) disulfide bond. We will also highlight application of the isoacyl concept to address the hydrophobicity of the A-chain and preparative recovery of both chains. Application of the isoacyl route afforded human insulin in 24% overall yield based on initial resin substitution representing the most efficient chemical synthesis of human insulin to date.