Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is making inroads into clinical laboratories. These instruments are most often used to perform laboratory-developed tests (LDT), but their operation is impeded by inexperienced personnel, complex hardware and software, the components of which may be produced by multiple manufacturers, and by a lack of literature that would guide the users in maintaining them. Although regular maintenance is essential for optimal continuous use of the LC-MS/MS system and methods, instrument and method maintenance may be ignored, leading to extended downtime, reduced productivity, suboptimal quality of results, or even instrument breakdown. Instrument manufacturers may provide information on maintenance of their hardware, but they don’t have instructions on developing a comprehensive maintenance plan that would cover the whole LC-MS/MS testing system. This publication is intended to help laboratory personnel and directors establish a comprehensive maintenance program for their LC-MS/MS instruments and methods. The maintenance needs of each component of an LC-MS/MS, the liquid chromatograph, mass spectrometer, computer, and methods are discussed, and information on possible maintenance actions and direction on developing a maintenance plan is provided with practical examples of maintenance logs.