We carried out an iTRAQ-based quantitative proteomic analysis of gallbladder cancer and adjacent non-tumor tissue to systematically identify differentially expressed proteins in gallbladder cancer. Ten gallbladder adenocarcinoma and ten adjacent non-tumor tissue samples were selected post pathological confirmation for the study. Samples were pooled and In-solution trypsin digestion was carried out. Post digestion, peptides were iTRAQ labeled with 114 and 115 (gallbladder adenocarcinoma) and 116 and 117 (adjacent non-tumor samples). LC-MS/MS analysis of SCX fractions was carried out using a reversed phase analytical C18 column connected to 1200 Series Nanoflow LC interfaced with LTQ-Orbitrap Velos. Data were acquired using Xcalibur 2.1. Proteome Discoverer (v 1.3) suite was used for quantitation and database searches. LC-MS/MS data were searched using Mascot and SEQUEST search algorithms against Human RefSeq 50 supplemented with frequently observed contaminants.