Mitochondria are critical organelles for energy production in eukaryotic cells, and their functionality is crucial for cell survival, especially under stress conditions (Cui et al, Fungal Genet Biol 103:42–54, 2017). In fungi, mitochondrial function can be assessed by monitoring the ability of yeast cells to grow on non-fermentable carbon sources (NFCs) such as glycerol or ethanol instead of carbon, which require functional mitochondria for energy production. This protocol describes the method for assessing mitochondrial health by growing yeast on agar plates with one of the NFCs instead of fermentable carbon. Cells with defective mitochondria show impaired growth on these NFCs, as they cannot efficiently generate energy through oxidative phosphorylation. In contrast, yeast with healthy mitochondria can grow well in the presence of these carbon sources. The protocol begins by growing yeast cells in a standard-rich medium to establish a starting population. The yeast cells are inoculated onto agar plates containing NFCs, such as glycerol, ethanol or lactate to assess their ability to utilize these substrates for growth (Smith et al, Endocr Rev 39:489–517, 2018; Zeng et al, Front Cell Infect Microbiol 13. https://doi.org/10.3389/fcimb.2023.1136698, 2023). The principle behind this assay is that NFCs require functioning mitochondrial respiration for energy production, as yeast cells can no longer rely on glycolysis alone. If the mitochondria are defective, the cells cannot efficiently metabolize these carbon sources and show poor or no growth on the plates. In contrast, wild-type yeasts with functioning mitochondria can grow vigorously. After inoculation, the plates are incubated at an optimal temperature (usually 30 °C) and the growth patterns are observed. The extent of growth is compared between different carbon sources and provides information about the health and functionality of the mitochondria. This method is widely used in mitochondrial research and genetic studies related to mitochondrial defects. This chapter provides a detailed protocol for testing mitochondrial function in yeast cells using NFCs and provides a reliable, reproducible assay for mitochondrial health and viability.