Phosphorus-31 magnetic resonance spectroscopy (31P-MRS) is a non-invasive method that provides useful information about metabolism and phosphoenergetic status in both physiologic and pathologic conditions of the human brain. With the progressive advances in magnetic resonance imaging (MRI) technology, particularly with higher magnetic field strengths, 31P-MRS has been more easily implemented and more readily available in the past few years, which has increasingly extended its access and favored its use in different research fields. However, the current knowledge about this advanced neuroimaging modality is still scarce and fragmented in the literature. Hence, in order to contribute to future researches and to shorten the gap between neuroscientific studies and common clinical routines, we present a comprehensive review about the basic technical aspects and biomedical applications of 31P-MRS.v