Adoptive cell therapy has become an important component of virus-refraction-treatment after allogeneic stem cell transplantations (ASCT). Enabling direct visualization and efficient enrichment of antigen-specific cytotoxic CD8+ T-Lymphocytes (CTLs) Streptamer technology may allow the identification of rare tumor-(WT1)-antigen specific CTLs as an option for treatment of AML-relapse-patients. To examine quality and sensitivity of Streptamer technology, a defined number of CMV pp65 A*0201 (NLVPMVATV) specific CTLs were titrated in blood without CMV-specific CTLs. Single-platform flow cytometry and True Count tubes provided the benefit to assess absolute cell numbers directly within the blood samples. Intra-assay-deviation was calculated after application of CMV pp65 B*0702 (TPRVTGGGAM) Streptamers in ten separate stainings, using the same donor-material. For WT1 donor-screening, we used a HLA A*0201 restricted Streptamer, presenting the peptide RMFPNAPYL. Quality control through spiking of antigen-specific CTLs showed a linearity of r2>0.99 and a detection limit of 1 CMV pp65-specific CTL per microliter, correlating with a frequency of 0.032±0.0033. Intra-assay-deviation stated a frequency of 0.6±0.03 (SD 5%). Screening results for WT-1 specific CTLs in 88 HLA A*0201 positive donors with a mean frequency of 0.003 and a SD of 0.005 corresponded to background noise. Also the maximum frequency of 0.026±0.005 did not state a distinct population. In comparison CMV A*0201 pp65/B*0702 pp65 screening in the same group of donors showed a mean value of 0.219±0.899/0.784±1.602 and a maximum frequency of 7.596±0.899/5.175±1.602. In conclusion, Streptamer technology is a reliable technique with high sensitivity and reproducibility. Also, our data indicate that the frequency of WT-1 specific CTLs among healthy donors is below the detection limit of Streptamer technology.