The advent of recombinant DNA technologies in the last two decades has made it possible to synthesize practically unlimited amounts of human proteins for therapeutic usage. This synthetic approach has been particularly valuable in the case of the pituitary hormones, growth hormone, prolactin, thyrotropin, luteinizing hormone and folliclestimulating hormone, which have important diagnostic and therapeutic applications. To determine the identity, purity, potency, safety and stability of these synthetic proteins, a variety of analytical techniques have been recommended, which are mostly based on physico-chemical properties of the proteins. Among these techniques High Performance Liquid Chromatography (HPLC), exploiting differences in protein properties such as size, hydrophobicity and charge, has become increasingly useful. Its accuracy, sensitivity, resolving power, speed and versatility have not only been established in research, but also in the quality and process control of industrial protein synthesis. In the case of biologically active proteins such as hormones, it has become more and more evident that HPLC is now strongly complementing, if not substituting, the classical biological and immunological assays whose accuracy limitations are well known. Moreover, it has been shown that the potential for early detection of impurities and molecular alterations provided by this technique allows more efficient and effective control both at the beginning and along the manufacturing process, leading to improved and more economical end products. This review article describes and analyzes recent developments of the main HPLC modalities, applied in the analysis and characterization of the above mentioned hormones, especially as they are relevant to the biopharmaceutical industry.