Cancer vaccines represent a promising strategy in tumor immunotherapy, aiming to elicit robust immune responses against tumor-specific antigens. The accurate measurement of antibody responses is critical in evaluating vaccine efficacy. Enzyme-linked immunosorbent assay (ELISA) offers a reliable, sensitive, and specific method for this purpose. This chapter discusses the development and optimization of ELISA for detecting total and antigen-specific antibodies in cancer vaccine research. Key considerations include customization of ELISA protocols, optimization of assay parameters, and selection of appropriate buffer systems, substrates, and controls. By standardizing these elements, researchers can achieve consistent and reproducible results, advancing the field of cancer immunotherapy and enhancing our understanding of immune mechanisms in oncology.