Lentiviral vectors are being used in a growing number of clinical applications, including T cell immunotherapy for cancer. As this new technology moves forward, a safety concern is the inadvertent recombination and subsequent development of a replication-competent lentivirus (RCL) during the manufacture of the vector material. To assess this risk, regulators have required screening of T cell products infused into patients for RCL. Since vector particles have many of the proteins and nucleotide sequences found in RCL, a biologic assay has proven the most sensitive method for RCL detection. As regulators have required screening of up to 108 cells per T cell product, this method described a procedure for assessing RCL contamination of large-volume T cell products.