Monitoring viral infections of cell cultures is largely neglected although the viruses may have an impact on the physiology of cells and may constitute a biohazard regarding laboratory safety and safety of bioactive agents produced by cell cultures. PCR, immunological assays, and enzyme activity tests represent common methods to detect virus infections. We have screened more than 300 Cancer Cell Line Encyclopedia RNA sequencing and 60 whole exome sequencing human cell lines data sets for specific viral sequences and general viral nucleotide and protein sequence assessment applying the Taxonomer bioinformatics tool developed by IDbyDNA. The results were compared with our previous findings from virus specific PCR analyses. Both, the results obtained from the direct alignment method and the Taxonomer alignment method revealed a complete concordance with the PCR results: twenty cell lines were found to be infected with five virus species. Taxonomer further uncovered a bovine polyomavirus infection in the breast cancer cell line SK-BR-3 most likely introduced by contaminated fetal bovine serum. RNA-Seq data sets were more sensitive for virus detection although a significant proportion of cell lines revealed low numbers of virus specific alignments attributable to low level nucleotide contamination during RNA preparation or sequencing procedure. Low quality reads leading to Taxonomer false positive results can be eliminated by trimming the sequence data before analysis. One further important result is that no viruses were detected that had never been shown to occur in cell cultures. The results prove that the currently applied testing of cell cultures is adequate for the detection of contamination and for the risk assessment of cell cultures. The results emphasize that next generation sequencing is an efficient tool to determine the viral infection status of human cells.