L-Carnitine and its esters are products of intermediary metabolism of organisms. The distribution pattern or the favored excretion of individual acylcarnitines tells something about metabolic diseases. The determination of the urinary acylcarnitine pattern by flow injection analysis (FIA)-electrospray ionization (ESI)-mass spectrometry (MS) is presented. Groups of healthy probands and patients suffering from diabetes mellitus were investigated due to their significant acylcarnitine profile. The statistical analysis of data sets obtained clearly shows a difference in the acylcarnitine pattern of healthy and sick probands. In comparison to the controls, diabetes mellitus patients excrete more long-chain carnitine esters ranging from dodecanoyl to palmitoylcarnitine. Thus, the urinary acylcarnitine pattern determined by ESI-MS can be a useful tool in the diagnosis and therapy monitoring of diabetes mellitus.