Therapeutic genetically modified vaccines administered to patients with cancer are expected to induce and sustain tumor-specific immune responses. To measure these responses, monitoring strategies including a range of antibody-based and cellular assays have been developed. Single-cell cellular assays capable of detecting fewer than 1/10,000 of antigen-specific T lymphocytes in the tested population are currently in demand. ELISPOT assay, cytokine flow cytometry, and tetramer binding were recently compared by us in the context of monitoring of a dendritic cell-based multi-peptide vaccination trial in patients with metastatic melanoma. These single-cell assays were not found to be concordant in measurements of the frequency of peptide-specific T cells or in detecting pre- to post-vaccine changes in the frequency of these T cells. Implications of the results for monitoring of genetically modified vaccines are discussed, and the plan for a rational approach to monitoring and for selection of monitoring assays is proposed. Monitoring of human gene therapy or vaccination trials in patients with advanced cancers, who are often immunocompromised and thus poorly responsive, is a challenge. This challenge is best met by utilizing experienced reference laboratories for immune monitoring of clinical trials.