Creatine is produced by the body and plays a major role in recycling ATP. It also stabilizes pH in certain tissues. Creatine can be analyzed by this reverse phase (RP) HPLC method with simple conditions. The mobile phase contains acetonitrile (MeCN), water, and phosphoric acid. For Mass-Spec (MS) compatible applications the phosphoric acid needs to be replaced with formic acid. Smaller 3 µm particle columns are available for fast UPLC applications. This liquid chromatography method is scalable and can be used for isolation of impurities in preparative separation. It also suitable for pharmacokinetics.