The prevailing view is that not only can some of the tumor antigens be used as biosensors for cancers, but also they may indeed be used as targets for immunotherapy. The identification of tumor antigens becomes a vital step in oncology research. Both the humoral immune system and the cellular immune system are activated in response to a tumor antigen in vivo of patients with tumor. Immune effector molecules and cells can be used to screen and identify tumor antigens. Specific T cells, including CD8+ and CD4+ T cells, can identify T cell epitopes, and specific antibodies in sera can identify B cell epitopes. The researchers have studied this area for decades. Initially, they explored tumor antigens with the use of 1-D SDS–PAGE and sandwich ELISAs. Since 1990s, CTL screening approach and peptide elution approach had been established. After that, SEREX, SERPA and protein microarray technology have become the mainstream highthroughput strategies for identifying tumor antigens. There are some other approaches, such as combinatorial peptide libraries, representational difference analysis of cDNA and bioinformatics methods. This review’s aim is to describe the generation, the theory, the key protocols and the application of some main techniques and provide their benefits and drawbacks.