Hyaluronic acid is a glycosaminoglycan, which is one of the main components of the extracellular matrix, contributes significantly to cell proliferation and migration, and is involved in many physiological and pathological biologic processes, such as the progression of some malignant tumors. Therefore, the determination of its amounts may be of use for monitoring the progress of some diseases and/or as prognostic/diagnostic marker. Hyaluronan has been used for the therapy of osteoarthritis, for ophthalmic and cosmetic surgeries, and is under investigation for numerous other diseases. In this review, after a short introduction to hyaluronan structure and biologic roles, the electrophoretic, chromatographic and solid-phase assays used for determination of its concentration in biologic samples and in drug formulations are presented.