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Objective: The aim of this study is to compare markers found in wound fluid of healing and non-healing diabetic foot ulcers using tandem mass spectrometry. Methods: Wound fluid samples were collected using a microfilament, centrifugated and frozen at -80C. Clinical outcome of the ulcers was determined after 6 months follow-up. Proteomic analysis of the samples was conducted and compared between 5 healing and 3 non-healing ulcers using the Fisher’s exact test with Benjamini-Hochberg’s corrected P values. Pathway-specific analysis was done to examine the functional differences in up- and downregulated proteins. Results: Up to 2155 proteins were identified in each sample, 113 of which were differentially expressed between two groups. Proteins upregulated in non-healing wounds were mainly associated with neutrophil degranulation, inflammatory/immune response, and cell adhesion and migration. The downregulated proteins were linked to smooth muscle cell proliferation, regulation of gene expression, and Wnt signaling pathway, proteins regulated by insulin and glucose and most of the proteins involved in negative regulation of apoptosis. Conclusions: Our findings signal activation of naive inflammatory pathway and cell migration, and potential downregulation of gene transcription and expression, cytoskeleton and intracellular calcium control mechanisms in non-healing diabetic ulcers. Disclosure N. Vatankhah: None. V. Jalili: None. Y. Jahangiri: None.