Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzyme for the synthesis of glycosphingolipids. Due to its diverse roles in physiology and diseases, GCS may be a disease marker and drug target. Current assays for enzymes including GCS are based on reactions conducted in a test tube using enzyme preparations. Measurement of enzyme activity in laboratory-made conditions cannot directly evaluate the role of GCS in cells. Here, we introduce a new approach to determine GCS cellular activity using fluorescent NBD C6-ceramide in vivo. Cellular GCS transfers UDP-glucose to NBD C6-ceramide and produces NBD C6-glucosylceramide. C6-glucosylceramide is then separated from C6-ceramide by thin-layer chromatography and both are then quantitated by spectrophotometer. This cell-based method is able to quantitate glucosylceramide in pmol range, produced by approximately 50,000 cells or 1.0 mg tissue. This method has been used successfully to evaluate the degrees of GCS enzyme in cells and in tumors subjected to gene manipulation and chemical inhibition. These data indicate that this cell-based fluorescent method is direct, reproducible, and simple for assessing ceramide glycosylation. It is applicable to validate GCS activity in drug-resistant cancers and in other disorders.