Chemical cytometry employs modern analytical methods to study the differences in composition between single cells to better understand development, cellular differentiation, and disease. Metabolic cytometry is a form of chemical cytometry wherein cells are incubated with and allowed to metabolize fluorescently labeled small molecules. Capillary electrophoresis with laser-induced fluorescence detection is then used to characterize the extent of metabolism at the single cell level. To date, all metabolic cytometry experiments have used conventional two-dimensional cell cultures. HCT 116 spheroids are a three-dimensional cell culture system, morphologically and phenotypically similar to tumors. Here, intact HCT 116 multicellular spheroids were simultaneously incubated with three fluorescently labeled glycosphingolipid substrates, GM3-BODIPY-FL, GM1-BODIPY-TMR, and lactosylceramide-BODIPY-650/665. These substrates are spectrally distinct, and their use allows the simultaneous probing of metabolism at three different points in the glycolipid metabolic cascade. Beginning with intact spheroids, a serial trypsinization and trituration procedure was used to isolate single cells from spatially distinct regions of the spheroid. Cells from the distinct regions showed unique metabolic patterns. Treatment with the lysosomal inhibitor and potential chemotherapeutic chloroquine consistently decreased catabolism for all substrates. Nearly 200 cells were taken for analysis. Principal component analysis with a multivariate measure of precision was used to quantify cell-to-cell variability in glycosphingolipid metabolism as a function of cellular localization and chloroquine treatment. While cells from different regions exhibited differences in metabolism, the heterogeneity in metabolism did not differ significantly across the experimental conditions.