Ceramide is a bioactive lipid involved in the induction of apoptosis and is the precursor to several sphingolipids, including sphingomyelin, the gangliosides, and sphingosine. Ceramide production is increased in response to stress and toxic agents. Because modulation of ceramide levels has been shown to affect sensitivity and/or resistance to therapeutic agents, it will be important to assess the activity of sphingolipid metabolic pathways when investigating the mode of action of antitumor drugs. This chapter summarizes protocols for quantitating the level of apoptosis, the activities of acidic sphingomyelinase, neutral sphingomyelinase, glycosylceramide synthase, sphingomyelin synthase, and ceramidase, and the amount of ceramide in tumor xenografts in nude mice.