Lung cancer is the leading cause of cancer-related to death in both men and women. Protein biomarkers for lung cancer were investigated using the expression of proteins from lung cancer cell line (A549) and compared with those of normal lung fibroblast cell line (MRC-5). Two-dimensional gel electrophoresis of A549 and MRC-5 cells was carried out and followed by protein identification using nanoelectrospray tandem mass spectrometry. Most proteins over expressed in A549 cells were phosphoproteins such as lamin AC 70 kDa, aldehyde dehydrogenase, alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and peroxiredoxin. Moreover, some proteins were expressed only in A549 cells such as heterogeneous ribonucleoprotein A1, nuclear corepressor KAP1, transketolase and cytokeratin 18. Furthermore, the phosphoprotein chaperonin 60 was highly expressed in A549 cells. It is known to function in protein interactions and protein conformation. The over expression of this protein in cells may result in abnormalities of protein conformation and lead to early stage cancer. These proteins may be used as biomarker of lung cancer for early detection and clinical prognosis.