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The use of high-performance liquid chromatography combined with mass spectrometry (HPLC-MS) or tandem mass spectrometry (HPLC-MS-MS) has proven to be the analytical technique of choice for most assays used in various stages of new drug discovery. A summary of the key components of HPLC-MS systems, as well as an overview of major application areas that use this technique as part of the drug discovery process, will be described here. This review will also provide an introduction into the various types of mass spectrometers that can be selected for the multiple tasks that can be performed using LC-MS as the analytical tool. The strategies for optimizing the use of this technique and also the potential problems and how to avoid them will be highlighted.