Background: Chimeric antigen receptor (CAR) T cell therapy is highly effective in treating hematologic malignancies, and major efforts are being made to achieve similar efficacy in solid tumors. The greater potency of CAR-T cells compared to antibody therapeutics demands a more stringent CAR-T target safety assessment to avoid adverse events resulting from “on-target/off-tumor” activity. Furthermore, it is critical to track and monitor CAR+ T cells within intact tissue and tumor to understand the mechanisms underlying off-tumor toxicity and efficacy in tumor killing. Methods: We employed the RNAscope in situ hybridization (ISH) technology to assess target expression specificity and to track CAR-T cell distribution and activation in xenograft and host tissues using the RPMI-8226 xenograft mouse model. Results: For the CAR-T target candidates, Target X and Target Y, RNA ISH revealed that Target X was only expressed in the xenograft tumor and in no mouse organs, while Target Y was found to be expressed at low levels in mouse lung and liver, as well as in the xenograft tumor. Duplex RNA ISH assays with probes targeting the CAR 3’ UTR and either IFNG or GZMB allowed for highly sensitive and specific detection of CAR-T cells and their activation state in both tumor and normal tissues from vehicle, Target X CAR-T cell, or Target Y CAR-T cell treated mice. Activated Target X CAR-T cells expressing GZMB and IFNG were found only in the xenograft tumor, where Target X was expressed. In contrast, activated Target Y CAR-T cells were found almost exclusively in mouse lung and liver, with very few Target Y CAR-T cells being found in the xenograft tumor. Lastly, a multiplex ISH-IHC approach confirmed the presence of activated Target X CAR-T cells in the xenograft tumor through simultaneous detection of the Target X CAR 3’ UTR, IFNG, GZMB, and CD3. Conclusions: These data demonstrate how the RNAscope assay can be utilized for CAR-T cell efficacy and safety/toxicity assessment in preclinical models by detecting very low levels of target antigen expression in off-tumor tissues and monitoring CAR-T cell pharmacodynamics and activation in tumor models and can also be applied for assessing TCR-T cell activity in tumors.