A better understanding of immune effector and regulatory pathways has led to innovative, and complex, immunotherapy strategies. CD8(+) cytolytic T lymphocytes (CTL) provide one common pathway of tumor cell destruction. The peripheral blood CTL compartment typically comprises a minority of anti-tumor CD8(+) lymphocytes and the determination of their number during clinical trials is the focus of various laboratory methods. We have monitored tumor specific CD8(+) as well as CD4(+) lymphocyte precursor frequencies in the peripheral blood using a Dye Dilution Proliferation Assay (DDPA). We summarize our experience applying DDPA in a multi-parameter, antigen-specific assay, detailing some of its complexities and advantages. We provide examples of our clinical trial results showing tumor-specific CD8(+) and CD4(+) precursor frequency (PF) data in patients being treated on novel immunotherapy trials.