Preparative high-performance liquid chromatography (HPLC) has become a mainstay of natural product isolation and purification. The various modes available (e.g., normal-phase, reversed-phase, size exclusion, and ion-exchange) to date can be used to purify most classes of natural products. This chapter presents an overview of the different modes along with a practical guide as to how to purify a natural product using the most robust and widely used of the modes, namely reversed-phase preparative HPLC. Instrumentation setup and detection methods, sample preparation, method development, and sample work up are also discussed.