Mitochondria are essential for several biological processes including energy metabolism and cell survival. Accordingly, impaired mitochondrial function is involved in a wide range of human pathologies including diabetes, cancer, cardiovascular, and neurodegenerative diseases. Within the past decade a growing body of evidence indicates that reversible phosphorylation plays an important role in the regulation of a variety of mitochondrial processes as well as tissue-specific mitochondrial functions in mammals. The rapidly increasing number of mitochondrial phosphorylation sites and phosphoproteins identified is largely ascribed to recent advances in phosphoproteomic technologies such as fractionation, phosphopeptide enrichment, and high-sensitivity mass spectrometry. However, the functional importance and the specific kinases and phosphatases involved have yet to be determined for the majority of these mitochondrial phosphorylation sites. This review summarizes the progress in establishing the mammalian mitochondrial phosphoproteome and the technical challenges encountered while characterizing it, with a particular focus on large-scale phosphoproteomic studies of mitochondria from human skeletal muscle.