Preparative HPLC (prep-HPLC) has emerged as a mainstay of natural products isolation and purification. The various modes available to date, e.g. normal phase, reversed-phase, size exclusion, and ion exchange, 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 prep-HPLC. Instrumentation set-up and detection methods, sample preparation, method development, and sample work up are also discussed.