High-performance liquid chromatography (HPLC) is a refinement of traditional column chromatographic techniques. The speed of analysis and the resolution are increased with new column-packing materials and eluant pumped through the column at high pressure. The potential for achieving measurements of hormones in small volumes of plasma or urine is limited, both in normal and pathological situations. Using HPLC with ultraviolet absorption, the detection limit is only nanogram amounts of hormones per milliliter of blood serum. The applications of the technique to specific hormones from recent and older literature will be used throughout this chapter to illustrate aspects of the technology.