Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Currently, different techniques are applied for CER quantitation, some of which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 microg of cell protein corresponding to about 20, 000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research.