Abstract: The development of cell therapy has focused on providing novel therapeutic cell-based products to treat various diseases. Chimeric antigen receptor (CAR)-T cell therapy is one of the novel cellular therapeutic options for cancer patients, such as the B-cell malignancies. To effectively develop and manufacture CAR-T products, it is imperative to assess the critical quality attributes (CQAs) of CAR-T cells, which is required by the chemistry manufacturing and controls (CMC) criteria standards as well as suggested by the recently published ISO guidance on cell characterization of cellular therapeutic products. Specifically, cell count and viability measurement are two of the main CQAs that are assessed during development, manufacturing, testing, and product release. In this work, we developed a rapid cell viability and apoptosis detection method to characterize the cell health and recovery of SubT1 cells after the plasmid-based electroporation process to create CAR expression cells using the Cellaca PLX Image Cytometer. Viability measurement of transduced SupT1 cells was performed by staining with acridine orange/propidium iodide (AO/PI). In addition, early and late-stage apoptosis were detected by staining transduced SupT1 cells with AnnexinV/PI and Caspase3/RubyDead, respectively. Results showed that the introduction of plasmids, other than the electroporation process itself, may induce 20 – 30% additional cell death in the process, indicated by viability results measured on Day 1 post transduction. Among all the AnnexinV or Caspase3 positive transduced SupT1 cells on Day 1 (22 – 34%), 70 - 80% of these cells were double positive in both AnnexinV and PI, or both Caspase3 and RubyDead, and only 20 – 30% of them were in the early-stage/late-stage apoptosis. Subsequently, the SupT1 cells were quickly recovered with their viabilities increased to >90% on Day 3. With such capability of rapid detection of cell viability and apoptosis, Cellaca PLX Image Cytometer can potentially support the characterization of factors that affects the quality of CAR-T cells in the manufacturing process, which may ultimately increase the success rate and reduce the waste for the development of CAR-T cell therapeutic products.