The levels and composition of sphingolipids and related metabolites are altered in aging and in common disorders such as diabetes and cancers, as well as in neurodegenerative, cardiovascular, and respiratory diseases. Changes in sphingolipids have been implicated as being an essential step in mitochondria-driven cell death. However, little is known about the precise sphingolipid composition and modulation in mitochondria or related organelles. Here, we used LC-MS/MS to analyze the presence of key components of the ceramide metabolic pathway in vivo and in vitro in purified ER, mitochondria-associated membranes (MAMs), and mitochondria. Specifically, we analyzed the sphingolipids in the three pathways that generate ceramide: sphinganine in the de novo ceramide pathway, SM in the breakdown pathway, and sphingosine in the salvage pathway. We observed sphingolipid profiles in mouse liver, mouse brain, and a human glioma cell line (U251). We analyzed the quantitative and qualitative changes of these sphingolipids during staurosporine-induced apoptosis in U251 cells. Ceramide (especially C16-ceramide) levels increased during early apoptosis possibly through a conversion from mitochondrial sphinganine and SM, but sphingosine and lactosyl- and glycosyl-ceramide levels were unaffected. We also found that ceramide generation is enhanced in mitochondria when SM levels are decreased in the MAM. This decrease was associated with an increase in acid sphingomyelinase activity in MAM. We conclude that meaningful sphingolipid modifications occur in MAM, the mitochondria, and the ER during the early steps of apoptosis.