The detailed analysis of lipids, especially phospholipids, has become progressively important, since these molecules represent intercellular messenger molecules and are involved in a number of diseases. Therefore, the analysis of the lipid pattern of blood, for example, may also be useful as a diagnostic tool. Unfortunately, suitable methods of phospholipid analysis are often time-consuming and tedious, since most of them include a variety of separation and derivatization steps. Methods allowing lipid analysis in a single step would be highly useful. The aim of this review is to show that high resolution 31P NMR spectroscopy is a convenient and precise analytical tool for the phospholipid analysis of extracts from biological samples (tissues and body fluids). The first part of this review discusses the physiological relevance of individual kinds of phospholipids and related, established analytical techniques for their investigation. The second part contains an overview on the requirements for 31P NMR to improve spectral resolution and sensitivity, its capabilities and its limitations. In the third part, we discuss the application of 31P NMR to the most important phospholipid classes, as well as different tissue and body fluid extracts. The article concludes with a brief look at future developments and potential applications of this technique.