In the context of ex vivo gene therapy or CAR-T cell therapy, vector copy number (VCN) analysis in transduced cells by lentiviral vectors enables the assessment of risk and therapeutic efficiency in patients. In this study, we show that a ddPCR-based method can replace easily a TaqMan qPCR assay for VCN analysis, by measuring the number of pro-viral DNA copies per host cell genome in blood samples. Firstly, we have identified key elements of sample preparation and set-up to further improve the performance characteristics of ddPCR method, resulting in an accurate analysis of VCN without the need for multiple replicates or an external calibrator, as required in qPCR methods. Secondly, we found that genomic DNA (gDNA) quantification by fluorometry allows a better prediction of the genomic copy number detected in ddPCR than by spectrophotometry. Then, we applied this new technology to analyze VCN in blood cells and also in colony-forming cells (CFC) derived from transduced CD34+ cells, using a NaOH cell lysis-based approach. Finally, we conclude that the multiplex ddPCR is able to analyze VCN more precisely than qPCR in all transduced hematopoietic cells, an assay useful for clinical applications.