High-performance liquid chromatography (HPLC) is a very powerful and versatile chromatographic technique for the separation of natural products (NPs) in complex matrices, such as crude extracts for selective detection and quantification or general profiling. The method is widespread and has been adapted to the analysis of a broad range of NPs generally without the need for complex sample preparation. The choice of the appropriate detection method in HPLC is crucial because of the diversity of NPs and the fact that there is no single technique for their efficient detection. In this review both qualitative and quantitative applications of HPLC with UV, DAD, FD, ECD, RID, FID, CL, ESLD, CAD, MS, MS-MS, and NMR are covered to provide a general, rather than an exhaustive, overview. The potential and limitations as well as some new trends in HPLC hyphenation are discussed.