Early detection and identification of oral pre‐malignancy or malignancy help in management of the disease and improve survival rates. Oral submucous fibrosis (OSMF) is a major threat to public health worldwide and especially in Southeast Asian countries. Identification of biomarkers is a necessary step toward early diagnosis and treatment. In this study, differentially expressed proteins between oral submucous fibrotic tissue and normal control tissues were recorded by proteomic analysis using two dimensional electrophoresis (2DE) and MALDI TOF mass spectrometry. By proteomic analysis, 15 proteins were found to be upregulated and 10 proteins downregulated in the OSMF tissues than the control tissues; among these identified proteins, Hsp‐70 1B, Calreticulin, and Lumican variant exhibited higher expression in OSMF tissues compared to the control tissues. Immunohistochemical analysis also showed elevated expression of these in OSMF tissues. Further validation was done by real time quantitative RT‐PCR analysis; gene expression of Hsp‐70 1B, Calreticulin, and Lumican variant were significantly increased (6.2‐, 3.3‐, 2.8‐ fold, respectively), whereas Enolase 1 was decreased by 0.5 fold in the OSMF tissues, consistent with proteomic results. The expression of proteins indicates that various cellular signaling pathways must be involved in the processes of fibrosis and suggests that expressed protein molecules play an important role in the pathogenesis of OSMF. These identified proteins may be potentially used in future studies of OSMF enabling to determine diagnostic marker or therapeutic targets of this precancerous condition of oral cavity.