Regulation of cellular metabolism is a central element governing the fate and function of T cells. However, the in vivo metabolic characteristics of rare cells, such as non-lymphoid tissue T cells, are poorly understood due to experimental limitations. Most techniques measuring cell metabolism require large cell numbers. The recent SCENITHTM method allows studying the metabolism of rare cells by flow cytometry. However, this technique requires cells to be isolated and cultured ex vivo, which may alter their metabolism. Here, we propose a new experimental approach, called in vivo SCENITH, to investigate the cellular metabolism of T cells in vivo at steady state in the spleen and lungs. For this purpose, we administered the metabolic modulators directly in mice, instead of applying these reagents ex vivo, as in the classical SCENITHTM method. Whereas ex vivo manipulation impacted viability and phenotype of T cells, this toxic effect was not observed in the in vivo SCENITH. We observed that conventional and regulatory T cells shared similar metabolic profiles. Importantly, whereas spleen T cells used both oxidative phosphorylation and glycolysis, the metabolism of T cell in the lungs was mainly based on oxidative phosphorylation. Finally, metabolic inhibitors that interfere with protein translation and energy availability downregulated Foxp3 expression in regulatory T cells. These results describe an expansion of SCENITHTM that allows to measure the metabolic profile of rare cells in vivo, revealing a high dependence on oxidative phosphorylation of lung T cells.