Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N-acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases.