Abstract: To develop a gel-based proteomic method to analyze the protein complexes in human pancreatic cancer cell line. Differential centrifugations and native PAGE were performed to separate the macromolecular fraction (containing protein complexes) in the whole cell lysate, followed by matrix-assisted laser desorption/ionization coupled with tandem time-of-flight mass spectrometric (MALDI-TOF/TOF MS) identifications. Two protein complexes were identified unambiguously by the combination of native PAGE and MS/MS, i.e., 20S proteasome (720 kDa) and Hsp90 dimmer (180 kDa). Native PAGE in conjunction with tandem mass spectrometry is simple, rapid and efficient method to study protein complexes.