beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.