Mitochondria may play important roles in metabolic diseases, neurodegeneration, and aging. Therefore, noninvasive methods to assess mitochondrial metabolic function in humans are urgently needed. We demonstrated that the ¹³C‐breath test using [1‐¹³C] acetate and [1‐¹³C] pyruvate holds great potential for assessing the metabolic status in mitochondria. The ¹³C‐breath test allows us to assess dynamic metabolic pathways noninvasively within 60 min. This test will not only facilitate clinical evaluation of patients but also provide huge opportunities for further understanding of these conditions. The potential of the ¹³C‐breath test using ¹³C‐labeled pyruvate in humans cannot be overemphasized.