Altered phospholipid compositions in skeletal muscle occur in muscular dystrophy and other pathological conditions; however, origins and relationships to the disease course are unclear. Liquid chromatography–mass spectrometry (LC–MS) technologies allow the determination of phospholipid compositions and alteration patterns in disease states. Here, we describe a basic protocol to prepare methanolic extracts from skeletal muscle, which may be analyzed by lipidomic LC–MS in order to detect compositional alterations in phosphatidylcholine, a major phospholipid species of skeletal muscle, as well as other phospholipid classes such as phosphatidylethanolamine. These analyses are useful to determine relative increases or decreases in individual phospholipid species between samples and capture changes in membrane compositions rather than the absolute quantities of lipids. LC–MS lipidomic analyses often yield large datasets containing hundreds of chromatographic peaks for each sample, and a ratiometric analysis strategy to determine the relative abundances of the major PC species is also briefly introduced.