The co-expression of CD4 and CD8 on a single mature T cell, known as a double-positive (DP) T cells, has been documented in peripheral tissues, particularly during viral infections and cancer. However, the function of DP T cells remains controversial. The study of DP T cells is challenging due to the propensity of single-positive CD8 and CD4 T cells to aggregate with one another, which may explain contradictory findings surrounding the function and phenotype of DP T cells. Here, we show that the use of Hoechst staining facilitates the differentiation of single DP T cells from CD8/CD4 T cell aggregates. The purity of Hoechstlow DP T cells isolated from spleen and lymph nodes (LNs) is consistently ~85.2 ± 0.8% and ~44.1 ± 1.3%, respectively. Knowing these purity factors allowed us to closely estimate the number of DP T cells in mice using conventional flow cytometry. Currently, DP T cells’ research is also limited by the scarcity of these cells in vivo. Therefore, we tested whether the injection of complexes of Interleukin-2 (IL-2) and the IL-2-specific monoclonal antibody S4B6, promotes the expansion of DPT cells. Indeed, IL-2/S4B6 complex injection significantly expanded the DP T cell numbers in the spleen. This methodology may allow for sufficient isolation of DP T cells for further investigation in vitro. In future, we will profile the transcriptomic signatures of DP T cells via single-cell RNA sequencing. Additionally, we will study the regulation of T cell receptor signalling and antigen recognition in DP T cells. We hypothesise that the close characterization of this underexplored T cell population will shed new light on its immunoregulatory properties and enhance our knowledge of how to develop more effective immunotherapies against diseases.