Objective:To establish a new approach to evaluate cellular enzyme activity of glucosylceramide synthase(GCS) accurately by fluorescent thin-layer chromatography and spectrophometer,and decide its role in drug-resistant cancer cells. Methods:Using fluorescent NBD C6-ceramide,GlcCer/Cer and ceramide glycosylation of drug-resistant breast cancer cell(MCF-7-AdrR)was measured under different concentrations of NBD C6-Ceramide and increasing treatment time. Radioenzymatic assay were performed on these cells as control method. Protein expressions of GCS and P-gp were assessed by Western-blot analysis and immuno-fluorescence staining. Results:These data indicated that 5 μmol/L of NBD-C6-Ceramide was the optimal concentration,and GCS activity increased with time delay. This method has been used to accurately evaluate the levels of GCS enzyme in drug-resistant cells,which is consistent with radioenzymatic assay. Ceramide glycosylation in MCF-7-adrR and MCF-7-adrR/GCS cells was dramatically increased compared with that in MCF-7 cells(P < 0.01),while the level of glycosylation in MCF-7-AdrR/asGCS was much lower than MCF-7-adrR(P < 0.01). Conclusion:The cell-based fluorescent method is direct,reproducible for assessing ceramide glycosylation. It was applicable to validate GCS activity in drug-resistant cancer cells and would further help to measure GCS activity in vivo.