Though the pancreatic islets are known to play critical roles in both Type 1 diabetes (T1D) and Type 2 diabetes (T2D), the distinct mechanisms are poorly understood. Recent findings demonstrate that chemical heterogeneity exists within the classic islet endocrine cell types, even in the normal/healthy pancreas setting. For example, there are subtypes of beta cells with distinct transcriptomes that respond differently to chemical signals. One critical scientific question is how does this cellular heterogeneity influence chemical signaling in T1D and T2D? We hypothesize that characteristic changes in chemical heterogeneity within the various human islet cell types during diabetes progression alter the signaling pathways inside and outside of the pancreatic islets to potentiate disease. Determining the dynamics of these changes will provide insight into chemical mechanisms that can be exploited for the prevention and treatment of T1D and T2D. Hence, there is a need for high-throughput and highly sensitive technologies capable of probing a sufficient number of chemical constituents in single cells. We propose three unique measurement platforms that implement advanced single cell mass spectrometry and analyte separation techniques to characterize the peptide and small-molecule content in individual human islet cells affected by T1D and T2D: 1) high-throughput single cell matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS); 2) quantitative capillary electrophoresis (CE)-MS with a focus on small molecule metabolites; and 3) CE-laser induced fluorescence (LIF) and liquid chromatography (LC)-MS characterization of the levels of D-amino acids. We expect that this research will dramatically improve the understanding of the cellular mechanisms of both T1D and T2D pathogenesis, and elucidate new chemical parameters characteristic of each disease, helping to identify novel pathways for therapeutic intervention.