The mechanisms driving progressive beta-cell dysfunction in type 2 diabetes (T2D) remain incompletely understood. This study aimed to identify pancreatic islet proteome changes that could predict diabetes onset. We isolated islets from non-diabetic subjects undergoing partial pancreatectomy, previously characterized for glucose tolerance, insulin sensitivity, and insulin secretion, using laser capture microdissection (LCM) and analyzed them via high-performance liquid chromatography-mass spectrometry (HPLC-MS). Proteomic analysis revealed that subjects with impaired glucose tolerance (IGT) had reductions in proteins regulating glycolysis (PGK1, G3P), lipid metabolism (ACBP, ARF1), glucose transport (14-3-3B), and insulin secretion (STARD10, CAPDS) compared to normal glucose tolerant (NGT) subjects. Additionally, IGT islets showed impaired expression of proteins involved in glucose- and incretin-stimulated insulin response (CREB1, IQGA1). Stratification by beta-cell glucose sensitivity (βGS) indicated that subjects with lower βGS exhibited reduced levels of insulin maturation (ERO1B) and anti-apoptotic proteins (CASP8, PAK2, SKP1), along with increased SEL1L, a factor promoting endocrine precursor differentiation. These findings suggest that early defects in glucose metabolism and insulin secretion characterize IGT, while reduced βGS may trigger compensatory mechanisms, through enhanced beta-cell survival or neogenesis, to delay T2D progression. Overall, proteomic alterations in prediabetic islets provide potential early predictive markers and targets for interventions aimed at preserving beta-cell function.