Chimeric antigen receptor (CAR)-T cell therapy is an emerging field for treatment of hematologic malignancies and other cancer types. To introduce new or corrected genes into the activated T-cells, lentiviral vectors are commonly used. Although lentiviral vectors are engineered to be replication defective, they potentially pose risks to human health, such as generation of a Replication Competent Lentivirus (RCL) capable of infecting non-target cells. qPCR assays for RCL monitoring are a rapid method to detect and usually quantify lentiviral genes, such as the envelope gene VSV-G (vesicular stomatitis virus G glycoprotein). Since Food and Drug Administration (FDA) guidance requires that all RCL positive results should be pursued by direct culture assay to obtain and characterize the infectious viral isolate, we concluded that a quantitative assay is not necessarily needed to detect RCL. Therefore, we developed an alternative approach for a highly sensitive, cost-efficient qualitative qPCR assay to assess the presence of the VSV-G sequence for the purpose of RCL monitoring.