The success of chimeric antigen receptor (CAR) T cell therapies for treating leukemia has resulted in a booming interest for the technology. Expression of a CAR in T cells allows redirection of their natural cytolytic activity toward cells presenting a specific designated surface antigen. Although CAR T cell therapies have thus far shown promising results mostly in B cell malignancy trials, interest in their potential to treat other diseases is on the rise, including using CAR T cells to control human immunodeficiency virus infection. The assessment of CAR T cell potency toward specific targets in vitro is a critical preclinical step. In this study, we describe novel assays that monitor the cytotoxicity of candidate CAR T cells toward simian immunodeficiency virus (SIV) infected CD4 T cells. The assays involve live cell imaging using a fluorescence microscopy system that records in real time the disappearance or appearance of targets infected with SIV carrying a fluorescent protein gene. The assays are highly reproducible, and their rapid turn around and reduced cost present a significant advance regarding the efficient preclinical evaluation of CAR T cell constructs and are broadly applicable to potential human diseases that could benefit from CAR T cell therapy.