During the manufacture of biopharmaceuticals, numerous adventitious agents have been detected in Master Cell Banks, end-of-production cells as well as bulk harvest fluid. Recently, a number of large-scale production bioreactors have become infected with Minute Virus of Mice (MVM) during cGMP (current good manufacturing practices) operations, and this has resulted in both the loss of product and the need for major cleaning validation procedures to be put in place. We have developed a simple DNA extraction/PCR assay to detect the presence of MVM in cell culture supernatant (conditioned cell fluids). This highly specific assay can detect 10 or fewer genome equivalents (copies) of MVM following PCR and gel electrophoresis visualization. For routine high-throughput detection, 300-100 copies could be consistently detected. The extraction procedure was shown to reliably detect MVM at a concentration of 1 TCID50/ml. The combination of the extraction/PCR procedure establishes a powerful, sensitive, specific assay that can detect the presence of MVM sequences with a 1-day turnaround time.