ABSTRACT We have developed a triplex TaqMan-based quantitative PCR assay for the human polyomaviruses JC (JCPyV) and BK (BKPyV). The assay simultaneously detects and quantifies both JCPyV and BKPyV in human clinical samples, and it includes an internal amplification control consisting of murine polyomavirus (MuPyV) plasmid DNA. We developed the assay for the Roche LightCycler 480 platform with the reporter dyes VIC, 6-FAM, and Cy5 for MuPyV, BKPyV, and JCPyV, respectively. The assay had a high specificity for BKPyV and JCPyV when either viral genome was present alone or in mixed samples over a range of 10 ¹ to 10 ⁷ copy numbers per reaction. The analytical sensitivity was 50 copies for BKPyV and 10 copies for JCPyV. The use of the MuPyV internal control ensured monitoring of the quality of the extraction and of PCR inhibition, even in samples such as cerebrospinal fluid and plasma in which controls based on host genes cannot be effectively used. In addition, we developed a similar assay using a different dye configuration (6-FAM, VIC, and NED) that could be used on an ABI 7500 Fast platform. This assay had sensitivities similar to those of the LightCycler 480 configuration for BKPyV and JCPyV when either viral genome was present alone, but the sensitivity of detection of BKPyV was greatly decreased when an excess of JCPyV (>100-fold) was present in the sample. This internally controlled combined assay offers greater convenience and cost-effectiveness compared to separate assays for each virus and can also detect unexpected PyV activations by testing for both viruses in all samples.