Current diagnostic and therapeutic strategies for lung cancer have had no significant impact on lung cancer mortality over the last several decades. This study used a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) discovery platform to generate protein expression profiles in search of overexpressed proteins in lung tumors as potentially novel molecular targets. Two differentially expressed protein peaks at m/z 12338 and 17882 in the MALDI-TOF spectra were identified in lung tumor specimens as macrophage migration inhibitory factor and cyclophilin A, respectively. Overexpression of both proteins was confirmed by Western blotting, and cyclophilin A was localized to the tumor cells by immunohistochemistry. These data demonstrate the feasibility of using a MALDI-TOF platform to generate protein expression profiles and identify potential molecular targets for cancer diagnostics and therapeutics.