Metabolic flexibility is vital for the cells to adapt to different energetic situations, allowing the organisms to adapt to changing conditions and survive challenges. One of the most important regulators of the metabolic flexibility is PGC-1α activity. PGC-1α integrates numerous signals and regulates a variety of transcription factors and nuclear receptors that together regulate mitochondrial homeostasis and fatty acid oxidation. One of the major ways that PGC-1α activity is regulated is by changes in its acetylation status. Thus measuring the acetylation status of PGC-1α is an important indicator of the metabolic flexibility of the cells. In this chapter, we describe an approach to evaluate PGC-1α acetylation in primary mouse myotubes. The method is applicable to other cell types and tissues as well.