Glycosphingolipids are structural components of mammalian cell membranes and are involved in essential cellular physiology such as cell-cell interaction, recognition, transmembrane signaling, proliferation and cell death. In this study, the simple quantitative method of monoglycoceramides-containing glucosylceramide and galactosylceramide was developed. The glycosylceramides extracted from culture cells and rat plasma were resolved by TLC, deacylated by SCDase and analyzed by HPLC-fluorescence detector at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. Limit of detection was approximately 0.1 pmol and limit of quantification was about 1 pmol for both monoglycoceramide standards. The recoveries of standard glucosylceramides from intra- and inter-day assays were 113.8 and 88.8% and those of galactosylceramides were 110.7 and 123.9%, respectively. The monoglycoceramide contents of SW-620 cells and rat plasma were $141.5{pm}5$ pmol/$1{ imes}10^6$ cells and $3.9{pm}0.3{mu}M$, respectively. The present analytical method provides a reproducible quantification and total content of monoglycoceramide which may be as a potential biomarker for lipid imbalance-related human diseases.