High-Performance Liquid Chromatography (HPLC) is a frequently utilized analytical method that has numerous characteristics, including high selectivity, sensitivity, and a reduced time requirement. Upon using this fabulous technique, the limit of detection is reduced, and the accuracy, precision, reliability, and specificity can be expanded. This technique plays a crucial role in both qualitative and quantitative analysis throughout the various stages of drug production, from the initial discovery of the drug to its excretion from the body. The role of High-Performance Liquid Chromatography (HPLC) in pharmaceutical analysis during various stages of drug discovery, production, and clinical use, including drug pharmacokinetics, is reviewed and discussed in detail. This provides an overview of the benefits of High-Performance Liquid Chromatography from the chemical, pharmaceutical, and clinical perspectives.