Glucosinolates (GLSs) are relatively inert (Z)-N-hydroximinosulfate esters, possessing a sulfur-linked β-D-glucopyranose moiety and a variable side chain, found almost exclusively in cruciferous vegetables. Following cell disruption, they are hydrolysed by plant myrosinases, forming a group of chemically reactive and biologically active compounds. There is considerable evidence that these breakdown products, when consumed in the diet, may affect the risk of developing chronic diseases. However, in order for any compound to exert an activity in vivo, it is necessary to reach the site of action in an appropriate form and sufficient concentration. Deleterious and toxic effects may be observed at high concentrations; hence, bioavailability is a key factor defining the physiological, beneficial dose window of GLS hydrolysis products (GLS-HPs). For some GLS-HPs, this window can be rather narrow; and therefore is a critical parameter to be considered. In this review we critically evaluate the present state of knowledge on all factors that affect bioavailability of GLS-HPs. This includes liberation from the plant material, absorption from the digestive system, distribution around the body, metabolism and excretion.