We sought to determine the differences with respect to the proteome of nasopharyngeal tissues between patients with nasopharyngeal carcinoma (NPC) and healthy controls by using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATHTM-MS) and ingenuity pathway analysis (IPA). Our primary purpose was to identify specific protein markers that can be applied for diagnosis or treatment of NPC.The CNE-1, CNE-2 and H1299 cell lines were cultured in stable isotope labeling of amino acids in cell culture (SILAC) medium for 10 generations to obtain labeled proteins. Thirty samples of NPC and 30 healthy control nasopharyngeal tissues were collected from the Department of Otolaryngology of the First Affiliated Hospital of Jinan University. Proteome of the nasopharyngeal tissues were analyzed and compared by SWATH-MS to identify differently expressed proteins. Further, extraction of target proteins and biological pathways was performed by IPA. Super-SILAC technique and liquid chromatography-tandem mass spectrometry were used to verify the reliability of the data obtained using SWATH-MS.We identified 1,415 differentially expressed proteins between NPC patients and healthy controls. On IPA analysis, EIF2AK2 and MAPK1 proteins were found to be enriched in multiple biological pathways and functional networks.The differentially expressed proteins EIF2AK2 and MAPK seem to play an important role in the biological network of NPC or may help discover the specific functional proteins of NPC. Further studies are required to identify the pathways and molecular mechanisms that underlie NPC.