Lentiviral vectors are useful vectors for stable transduction and permanent expression in dividing and non-dividing cells. In particular, third-generation lentiviral vectors have been engineered to be significantly safer than their second-generation counterparts, incorporating several safety features not present in earlier versions. For example, the tat gene, which is essential for the replication of wild-type human immunodeficiency virus type 1, has been deleted, and vector packaging functions have been distributed across three separate plasmids, further enhancing safety. In both research and clinical settings, having a reliable and accurate method for titering lentiviral vectors is critical. We have developed a method using the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element as a template for a real-time quantitative polymerase chain reaction, coupled with TRIzol lysis buffer for ribonucleic acid isolation. This method yielded results comparable to those from a commonly used commercial kit, offering advantages of speed, cost-effectiveness, and accuracy. It presents a viable, economical alternative for both research and clinical laboratories.