A new method for the simultaneous assay of D- and L-enantiomers of carnitine is described. The method is based on precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate [(+)FLEC] producing a diastereomeric derivative which can be detected both by UV absorbance and fluorescence detection. Also acyl esters of carnitine can be processed with this method, after alkaline hydrolysis. The D-enantiomer of carnitine and acylcarnitine can be detected at a concentration as low as 0.2% in the raw material and in pharmaceuticals. Assays can be carried out using an autoinjector either by HPLC or capillary electrophoresis (CE) because the derivative proved to be very stable. Its application is proposed for the routine assay of the enantiomeric excess of L-carnitine and their acyl esters in pharmaceutical products.