Virus contamination of com. valued cell cultures can be a health risk to the general population and imposes financial burdens on manufacturing and biopharmaceutical companies.We investigated the use of massively parallel sequencing/next-generation sequencing (MPS/NGS) and bioinformatics for the detection and subsequent identification of adventitious contamination.Specifically, the Illumina HiSeq 2000 instrument, in the 2 × 100 base pair (bp) paired end (PE) run configuration, was used to determine the identity and limit of detection of a DNA virus spiked into a virus vaccine, and an RNA virus spiked into a mammalian master cell bank (MCB).This configuration provided sufficient sequence read lengths and depth of coverage to detect and identify spiked-in SV40 and measles viruses in a background of vaccine, MCB, and host nucleic acid that make up the bulk of these samples.Furthermore, the detection of 30 SV40 reads within one tested sample suggested a < 1 plaque forming unit (pfu) sensitivity in a background of 2.8 × 107 infectious adenovirus serotype 5 (Ad5) particles.Results from subsequent virus vaccine testing suggested the presence of non-viable virus DNA contamination in the sample.Therefore, we propose that a multimodal approach, in which broad-range screening for known or unknown adventitious agents by MPS/NGS is complemented by targeted virus detection assays (e.g., PCR-based and infectivity assays), should provide the most useful safety monitoring information.We suggest that MPS/NGS and the accompanying bioinformatics is a sensitive, broad-range, and long-lasting tool with the ability to improve upon existing biosafety testing within a larger testing program.