Recent advances in multiplex immunohistochemistry techniques allow for quantitative, spatial identification of multiple immune parameters for enhanced diagnostic and prognostic insight. However, applying such techniques to murine fixed tissues, particularly sensitive epitopes, such as CD4, CD8α, and CD19, has been difficult. We compared different fixation protocols and Ag-retrieval techniques and validated the use of multiplex immunohistochemistry for detection of CD3+ CD4+ and CD3+ CD8+ T cell subsets in murine spleen and tumor. This allows for enumeration of these T cell subsets within immune environments, as well as the study of their spatial distribution.