Therapeutic vaccines administered to patients with cancer are expected to induce and sustain tumour-specific immune responses. However, anti-tumour responses are often compromised in such patients, and a new approach to evaluating their immune competence before vaccination is needed. Spontaneous apoptosis of activated anti-tumour effector cells, including CD8+ Tetramer+ T cells, occurring with a high frequency in the circulation of such patients may be one reason for low frequencies of peptide-specific T cells detectable after vaccination. For patients treated with anti-tumour vaccines, monitoring strategies, including a broad range of antibody-based and cellular assays, have been developed. Single-cell assays, using circulating PBMC and 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 (CFC) and tetramer binding were recently compared by us as part of a dendritic cell-based multi-peptide vaccination trial in patients with metastatic melanoma. All three assays performed under GLP conditions in an experienced monitoring laboratory detected a low frequency of CD8+ peptide-responsive T cells and were not found to be concordant in measuring pre- to post-vaccine changes in the frequency of these T cells. The selection of an assay for monitoring of anti-cancer vaccines is an important decision, which has to be undertaken rationally with a complete understanding of the assay limitations. The assay performance has to be validated in terms of sensitivity, specificity, inter- and intra-assay variability, and its coefficient of variation established. Performance of an assay with fresh vs. frozen/thawed specimens has to be evaluated carefully. The challenge of serial monitoring in patients receiving therapeutic vaccines is best met by using experienced reference laboratories.