Polyol dehydrogenases are enzymes that convert polyalcohols into sugars, using NAD+ as hydrogen acceptor. Sorbitol, mannitol or other polyalcohols can be used as substrates, and the activity of these enzymes is assessed by monitoring the production of NADH. This protocol was based on previously published methods (Maret and Auld, 1988; Fernández et al., 1993), and was used to evaluate polyol dehydrogenase activities in the digestive gland of gastropods. With the proper adaptations this procedure can be applied to other dehydrogenases and other organisms.