Unnoticed cell culture contamination by viruses, Mycoplasma, or other cell lines is not uncommon and a threat to laboratory safety and the quality of scientific results. We developed and validated a novel high-throughput Multiplex cell Contamination Test (McCT), which is currently able to detect 37 contamination markers in a single reaction. The assay is based on multiplex PCR with target-specific primers and subsequent hybridization of amplimers to specific oligonucleotide probes. McCT proved to be highly specific, sensitive and robust, and allows to analyze more than 1000 cell lysates per week. In conclusion, the novel McCT assay is a powerful high-throughput tool in assessing cell line purity.