Nearly one in ten Americans has type 2 diabetes mellitus (T2DM), a major source of morbidity, mortality, and healthcare costs. Most patients with diabetes are treated with medicines which may have adverse side effects. The prevalence of this disease necessitates the addition of new, safer compounds to the anti-diabetic arsenal. The recently reported Fatty Acid esters of Hydroxy Fatty Acids (FAHFAs) included eight Palmitic Acid esters of Hydroxy Stearic Acid (PAHSA) regioisomers, which may provide a new avenue for treatment of T2DM. In particular, 5- and 9-PAHSA increased insulin secretion, improved blood sugar, and decreased adipose tissue inflammation in diabetic mice. While this activity is encouraging, much remains unknown about PAHSAs, including the influence of chemical connectivity on their activity. Furthermore, the molecular targets of PAHSAs have not been fully elucidated. Until these questions are answered, there can be no hope of treating T2DM with an optimized PAHSA analog, nor means to rationally upregulate de novo PAHSA synthesis. This resulted in a scalable total synthesis of racemic 5- and 9-PAHSA, both enantiomers of 9-PAHSA, and preparation of a 30 membered library of PAHSA analogs to probe structure activity relationships. Furthermore, 6 chemical biology probes have been prepared to determine in an unbiased manner the molecular targets of PAHSAs and their metabolism.