Provided are methods of detecting replication competent virus, e.g., replication competent retrovirus such as gammaretrovirus or lentivirus, in a sample containing a cell transduced with a viral vector particle encoding a recombinant and or heterologous molecule, e.g., heterologous gene product. The methods may include assessing transcription of one or more target genes, such as viral genes, that are expressed in a retrovirus but not expressed in the viral vector particle. Replication competent retrovirus may be determined to be present if the levels of RNA of the one or more target genes is higher than a reference value, which can be measured directly or indirectly, including from a positive control sample containing RNA from the respective target gene at a known level and/or at or above the limit of detection of the assay.