BK virus (BKV) is a human polyomavirus which asymptomatically infects about 46-94% of the general population at childhood and remains latent in the kidneys after primary infection. Re-activation of BKV is recognised as a significant cause of renal allograft dysfunction and graft failure in renal transplant patients, and is linked to immunosuppressive regimens containing tacrolimus or mycophenolate mofetil. Management of BKV nephropathy involves reducing the dosage of immunosuppressants and/or antiviral treatment with cidofovir or leflunomide. Quantitation of BKV in blood and urine may help determine whether management therapy is effective by monitoring viral titres over time. To improve workflow, reliability and precision, the quantitative BKV-R-gene assay using the MagNAPure 96/LC480 (Roche Diagnostics) was evaluated against an in-house BKV real-time method (MagNAPure LC/LightCycler 1.0) for plasma and urine samples. Intra-assay and inter-assay precision was compared between the methods using 10 replicates of a 1.00 x106 copies/mL Zeptometrix NATtrol BK Virus External Control (Diagnostic Technology). Intra-assay percent coefficient of variation for BKV-R-gene and in-house BKV was 7.6% and 28.9% respectively. This equates to ±0.03 and ±0.18 log10 copies/mL respectively. A high degree of intra-assay precision was also noted for replicates of patient samples. Inter-assay precision assessed over 10 consecutive runs using the same Zeptometrix NATtrol BK Virus External Control was 39.0% and 71.3% respectively, corresponding to ±0.21 and ±0.29 log10 copies/mL. Assessment of linearity using a Zeptometrix NATtrol BK Virus Linearity Panel was also performed for the BK Virus R-gene Assay. Results for the linearity panel were concordant with the actual concentrations of BKV. Thirteen plasma and 57 urine samples were tested in a blinded prospective fashion over four consecutive runs using both assays and the results were compared. With the exception of 4 samples all samples tested correlated and were within ±1.0 log10. Overall the BK Virus R-gene assay performed well. This assay is well suited for routine use and can be used simultaneously with other R-gene assays.