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Enzyme-linked immunosorbent assay (ELISA) offers an effective, inexpensive, and reliable approach for the analysis of humoral immune responses. Here, we describe a protocol for measuring anti-fentanyl antibodies generated by the immunization of mice with novel opioid vaccine candidates. We describe steps for coating BSA-fentanyl antigen and standard wells. We then detail procedures for preparing buffers and performing assays. This protocol is adaptable for use with either BALB/c or C57BL/6J mice strains and can measure antibody isotypes to elucidate class switching. For complete details on the use and execution of this protocol, please refer to Stone et al..