An overview of the state-of-the art in HPLC coupled with fluorescence detection is presented. Over the last 20 years, the increasing number of methodological papers on this topic (4082 between 1994 and 2004 and 7725 between 2004 and 2014) is testament to its utility in bioanalytical applications. Compared with conventional UV absorbance detection used in HPLC, fluorescence detection can greatly enhance the sensitivity leading to limits of detection similar to those obtained with mass spectrometry, offering researchers a sensitive, robust and relatively inexpensive instrumental method. This work will focus on the analysis of pharmaceutical compounds in different biological matrices, either naturally fluorescent or derivatized with a fluorescent agent, and some of them chiral. Therapeutic applications, sample preparation and derivatization, sensitivity for each example are described.