Adoptive transfer of T-cells expressing anti-CD19 chimeric antigen receptors (CAR) has shown > 80% complete remission rates in acute B cell leukemias. However, therapeutic efficacy is low or absent for some other hematological malignancies and solid tumors, mostly due to limited CAR-T cell persistence and functional exhaustion post transplantation. It has been reported that anti-CD19 CAR-T cells sustain complete remission of leukemia and normal B cell aplasia even when their numbers in circulation are below the limit of detection by flow cytometry (FC) and detectable only by quantitative PCR (qPCR). Thus, developing a monitoring strategy that combines high sensitivity and multiparametric immunophenotypic evaluation is essential to predict, understand and improve the clinical response to CAR-T cell therapies. In this work, we aimed at developing a platform for monitoring the persistence of CAR-T cells using multiparametric FC and qPCR. To facilitate CAR-T cell tracking by FC in preclinical models, we developed a new lentiviral vector by cloning the enhanced green fluorescent protein (EGFP) gene downstream the anti-CD19 CAR gene. Lentiviral particles carrying the new construct were used to transduce primary T cells for CAR-T cell generation. FC analysis after transduction demonstrated that 44.8% of cells expressed CAR on the surface and that all CAR+ cells were also EGFP+, confirming the functionality of the new vector. Notably, the median fluorescence intensity of EGFP was 3.8-fold higher than that of CAR stained with an Alexa 647-conjagated antibody, even though this fluorophore has a high brightness index of 4. Thus, through EGFP fluorescence, this new construct allows direct CAR-T cell tracking by FC with a higher sensitivity compared to antibody staining. We next assessed the cytotoxicity of CAR19/GFP cells by coculturing them with Deep Red stained tumor cell lines either CD19+ (RAJI) or CD19- (K562) at a 1:1 effector to target ratio. After 16h of co-culture, FC data showed that CAR19/GFP cells eradicated 47% of CD19+ tumoral cells while sparing CD19- tumor cells, demonstrating that the CAR codified by this new vector is functional and CD19-specific. Lastly, we developed a CAR detection protocol based on qPCR. To that end, we designed a pair of specific primers and a probe spanning the costimulatory 4-1BB and CD3ζ domains of our anti-CD19 CAR. This primer/probe set was used to successfully detect CAR copies by qPCR in a plasmid standard curve ranging from 1010 to 1 CAR copy. This high sensitivity was accompanied by a high linearity (R2=0.99) and amplification efficiency (99.7%). To simulate a molecular test for CAR-T cell detection, we added 100 ng of genomic DNA to each point of the standard curve. This led to a slight reduction of sensitivity, allowing detection of at least 103 CAR copies (R2=0.99; amplification efficiency = 106.8%). Combined, this data demonstrates the generation of a fully functional CAR/EGFP reporter vector and the establishment of a protocol for molecular detection of CAR-T cells with high sensitivity by qPCR. This CAR-T cell-monitoring platform will provide invaluable data in preclinical and clinical studies on CAR-T cell persistence, a parameter intrinsically linked to therapeutic efficacy. Support: FAPESP grants 2013/08135-2, 2019/18672-1 and 2019/18702-8.