Translation initiation is the rate-limiting step of protein synthesis and highly regulated. Eukaryotic initiation factor 3 (eIF3) is the largest and most complex initiation factor consisting of 13 putative subunits. A growing number of studies suggest that eIF3 and its subunits may represent a new group of protooncogenes and associates with prognosis. They regulate translation of a subset of mRNAs involved in many cellular processes including proliferation, apoptosis, DNA repair, and cell cycle. Therefore, unveiling the mechanisms of eIF3 action in tumorigenesis may help identify attractive targets for cancer therapy. Here, we describe a series of methods used in the study of eIF3 function in regulating protein synthesis, tumorigenesis, and cellular response to therapeutic treatments.