Reactivation of Polyomavirus BKV is a severe complication in kidney transplant patients. Current treatment requires close monitoring, and modification of immunosuppressive drugs. As an important additional tool, the monitoring of BKV immunity has been based on detection of cytokine-secreting T cells upon BKV-antigen challenge. However, low frequent BKV-specific T cells are often barely detectable and their roles in BKV clearance remain unclear. Here, we analyzed the effects of immunosuppressive agents on BKV-specific T cells in vitro. Significant reductions in expression of several markers, and reduced killing functions upon treatment with calcineurin but not mTOR inhibitors were detected. However, effects of these drugs on expression of surface markers and GranzymeB were substantially less striking than effects on cytokine expression. Consequently, we applied a novel detection strategy for BKV-specific T cells in immunosuppressed kidney transplant patients using these more robust markers, and showed significantly improved sensitivity compared with the conventional IFNγ-based method. Using this strategy and 17-color flow cytometry, we found BKV-specific helper and cytolytic CD4+ T-cell subsets that differed in their memory phenotype, which corresponded with BKV clearance in kidney transplant patients. Thus, our results offer an improved detection strategy for BKV-specific T cells in kidney transplant patients, and shed light on the contributions of these cells to BKV clearance.