PURPOSE: Fibrovascular membrane (FVM), a pathological tissue causing retinal traction, represents an advanced stage of proliferative diabetic retinopathy (PDR). This study aimed to identify novel protein biomarkers associated with retinal microvascular damage leading to FVM formation and to evaluate the diagnostic potential of stratifin (SFN) and hornerin (HRNR) in patients with type 2 diabetes mellitus (T2DM) and diabetic retinopathy (DR). METHODS: Human retinal vasculature from non-diabetic donors and FVM tissues from PDR patients undergoing PPV were analyzed by one-dimensional liquid chromatography-mass spectrometry to identify differentially expressed proteins. Functional enrichment and protein interaction analyses were performed using STRING databases. Selected proteins' messenger RNA expression was quantified by quantitative PCR (qPCR) in peripheral blood mononuclear cells from healthy, T2DM, and DR with nephropathy (DR with DN) patients. Serum levels of SFN and HRNR were measured by ELISA in T2DM, DR, and DR with DN cohorts. Diagnostic performance was evaluated using receiver operating characteristic (ROC) curves. RESULTS: A total of 44 FVM specific proteins were identified, notably SFN and HRNR linked to retinal angiogenesis. Serum HRNR levels were significantly decreased in DR with DN compared with T2DM, whereas SFN levels were significantly increased in DR versus T2DM. ROC analyses demonstrated acceptable accuracy for differentiating DR stages based on serum SFN and HRNR levels. CONCLUSIONS: This is the first study to identify SFN and HRNR as systemic candidate biomarkers reflecting microvascular alterations in DR. Larger cohort studies are warranted to validate their clinical relevance for early detection and therapeutic targeting of DR.