A method for ceramide (CER) and diradylglycerol (DG) determination after normal-phase HPLC separation was developed. The free oxydril group of ceramide and diradylglycerol is coupled to the carboxylic group of the fluorescent label (+)-6-methoxy-α-methyl-2 naphthaleneacetic acid (NAP), using as catalytic agents 4-dimethylaminopyridine andN,N′-dicyclohexylcarbodiimide. The use of NAP-free acid instead of the halide-activated form ensures higher stability of the reagent, lower reaction temperatures, and improved yield and reproducibility. The yield of the reaction is greater than 90% after a period of 3 h at the temperature of −20°C. Over 85% of the starting material is recovered at the end of HPLC separation. The lower detection limit is below 5 ng for CER and 150 ng for DG. Under the conditions employed in the assay, no significant hydrolysis of triglycerides, sphingo- lipids, or phospholipids occurs and the esterification reaction is not affected by components of crude lipid extracts. Since separation and/or purification steps are not required, cellular levels of CER and DG can be easily and rapidly measured.