Obesity affects susceptibility to sexually transmitted diseases like genital her-pes, caused by herpes simplex virus (HSV) 2. The y8 T cells in the vagina play a major role in HSV-2 suppression. Here, we present a protocol for inducing HSV-2 infection intravaginally in high-fat diet-induced obese mice. We describe steps for isolating single cells from vaginal tissue and analyzing cells using sin-gle-cell RNA sequencing and flow cytometry. We then detail confirmation of the y8 T cell phenotype in vitro. For complete details on the use and execution of this protocol, please refer to Park et al.1