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Background The development of sensitive and reliable bioanalytical methods is critical for evaluating CAR T-cell expansion, persistence, and minimal residual disease (MRD) in clinical trials. DuoCAR20.19.22-D95 is an investigational autologous CAR T-cell therapy targeting CD19, CD20, and CD22, currently being evaluated in a Phase 1/2 trial in patients with relapsed or refractory B-cell malignancies. Here, we describe the validation of a flow cytometry-based assay focused on establishing the Limit of Blank (LOB), Limit of Detection (LOD), and Lower Limit of Quantitation (LLOQ) for detection of DuoCAR20.19.22-D95 CAR T cells in peripheral blood mononuclear cells (PBMCs). 3 reportables were assessed for LOB, LOD, and LLOQ – Reportable 8, 22, and 34.Methods Validation studies were conducted using cryopreserved PBMCs from three healthy donors. LOB and LOD were assessed using unspiked PBMCs stained with the full CAR T-cell detection panel and a gating control (FMX) control. LOB and LOD values were set at 1.645 SD and 3 SD above noise, respectively. For LLOQ determination, a DuoCAR20.19.22-D95 CAR T-cell product was spiked into PBMCs at a 1:5 ratio, followed by a seven-point, threefold serial dilution. All samples were stained with the CAR T-cell detection panel and acquired by flow cytometry. LLOQ was defined as the lowest concentration with ≥50 gated events and a coefficient of variation (%CV) ≤30% from the expected value. If the LOD exceeded this threshold, the LLOQ would be set to equal LOD.Results The LOB and LOD values were as follows: Time QC Gate/Non-Debris/Single Cells/Live/CD45+/CD3+/CD4+/CD19 CAR+ CD22 CAR+: LOB = 111.72 events; LOD & LLOQ = 144.99 events Time QC Gate/Non-Debris/Single Cells/Live/CD45+/CD3+/CD8+/CD19 CAR+ CD22 CAR+: LOB = 37.98 events; LOD & LLOQ = 41.54 events Time QC Gate/Non-Debris/Single Cells/Live/CD45+/CD3+/CD19 CAR+ CD22 CAR+: LOB = 159.53 events; LOD & LLOQ = 196.20 eventsFor all three reportables, the LLOQ corresponded to the calculated LOD, with all spike levels achieving ≥50 events and%CV ≤30%.Conclusions This validated flow cytometry assay demonstrates robust sensitivity and precision for detecting DuoCAR20.19.22-D95 CAR T cells in PBMC samples. The established LLOQ supports downstream pharmacokinetic, pharmacodynamic, and MRD assessments in the ongoing clinical trial. This assay enables sensitive monitoring of cellular kinetics, contributing to the overall evaluation of therapeutic safety and efficacy.